Figure One: Enzyme assay of yopH at different enzyme concentration amounts using a spectrophotometer read at 410 nm.
Figure Two: Graph of average absorbance read at 410 nm versus enzyme (yopH) concentration for the first trial.
Analysis: yopH surragote protein was used to run an enzyme assay and inhibition assay after it was properly expressed and purified. As more yopH was added to the solution more activity was oberserved because the solution turned more and more yellow after the addition of NaOH and there was an increase in absorbance measured for each solution with more yopH concentration. This being said the enzyme assay worked and the 10uL amount of yopH enzyme dilution will be used for inhibition assay because there is a significant jump from 5 uL to 10 uL and should properly be inhibited at this protein amount.
Inhibition Assay of yopH using 5852635 and 5250098:
Figure one: Inhibition assay of yopH against 5252635 inhibitor compound at different concentrations of the compound using a spectrophotometer read at 410 nm.
Figure Two: Graph of inhibition assay trial one using the inhibitory compound 5852635 against yopH. Absorbance at 410 nm versus inhibitor compound concentration.
Analysis: An inhibition assay was run with the yopH surrogate protein against a protein phosphatase inhibitor 5852635. When there was no concentration of inhibitor in the sample the absorbance reading was very similar to the absorbance of yopH at the 10 uL amount form the enzyme assay so this shows consistency which is good. Then once the inhibitor was addd to the solutions the activity of yopH greatly decreased. At the concentration of 0.6 for the inhibitor compound there was a slight increase in absorbance this could have been because at that high of a inhibitor concentration it is not optimal. The ortho positive control inhibitor is a good comparing point for the amount of activity that the protein should have in the presence of an inhibitor and because the activity is around the positive inhibitor absorbance this is really good! There was not a steady decrease like what was to be expected but this could be because at the inhibitor concentration at 0.1 it already inhibits the protein very well. This being said the next trial should be tested using even lower concentration of the inhibitor compound to see if this results in a more steady decrease.
Figure Three: Inhibition assay of yopH against 5250098 inhibitor compound at different inhibitor concentrations using a spectrophotometer read at 410 nm.
Figure Four: Graph of inhibition assay trial one using the inhibitory compound 5250098 against yopH. Absorbance at 410 nm versus inhibitor concentration.
Analysis: The same procedures were followed for this inhibition assay as the one that worked with the protein amount still at 10 uL, this timing using a different inhibitory compound ( 5250098). This compound did not inhibit the activity of yopH compared to the positive inhibitor. The samples where not inhibitor was added were consistent with the absorbance from the enzyme assay at 10 uL which is good, but the compound did not work to inhibit yopH activity. This is because the activity (read by absorbance) of the enzyme was not decreased. This could have been operator error or equipment error but both inhibition trials were run at the same time so that might not have been the case. Therefore this compound is not going to be used for further testing.
Virtual:
NIH library-
Table One: Top scoring ligand results from NIH library with the ligands docked into the active site of 4IL1 using GOLD.
Analysis: The NIH library, containing 446 ligands, was screened using GOLD and the top 46 scoring ligands are shown in the table above. There were two ligands that scored in the 90's which is really good and those two could potentially be good inhibitors of T. brucei protein phosphatase 2B. Also all 46 highest scoring ligands has really high scores as well which is good! The next step will be to compare these results to ICM results to see if there might be a good inhibitor.
HF9PlatesPlates Library-
Table Two: Top scoring ligand results form HF9PlatesPlates library with the ligands docked into the active site of 4IL1 using GOLD.
Analysis: The HF9PlatesPlates library, containing 400 ligands, was screened using GOLD and the top 40 scoring ligands are shown in the table above. These scores are not as good as the scores from the NIH library but the results are better than the CH306 library results. The few highest scoring ligands may or may not be good inhibitors but the next step will be to run this library through ICM and compare the results to see if there is a potentially good inhibitor.
Weeks 13 and 14
*week 13: tried expressing surrogate plates and found that the yopH plate was too old to use so I could not grow anything and the ftHAP plate was not expressing properly so new yopH plates were made and used for expression, purification and characterization.
Week 14:
Figure One: SDS-PAGE Gel of yopH surrogate protein after expression and purification. Lane two: Colorplus protein ladder. Lane three: sample one containing protein after large culture overnight (lysate after induction). Lane four: sample two containing protein pre-syringe filtration (soluble fraction). Lane five: sample three containing flow through. Lane six: sample four containing wash. Lane seven: sample five containing first elution. Lane eight: sample six containing second elution.
Analysis: Expression, purification and characterization finally worked for yopH!! Option A was followed with the normal procedures for expression and purification, and the normal lysis buffer was used. Elution one does not have much contamination so FPLC can be optional. May try FPLC with only have of the elution one solution that way the protein is not lost during FPLC and there will still be some to do enzyme assays with.
Virtual on my target: T. brucei protein phosphatase 2B
Table One: Results of positive and negative controls ligands docked into the active site of the homology model of 4IL1.
Analysis: The top three highest scores were 141.88, 129.95, and 121.29 all of which are positive control ligands which is a good thing because a high score for a negative control ligand would mean something is wrong with the defined active site. Because there were three positive ligands with high scores now 4IL1 can be screened against several libraries and compare the results with the three high scoring positive ligands to determine if any novel ligands can be found that would inhibit T. brucei protein phosphatase 2B, even though enzyme assays and inhibition assays will not be run with this protein.
CH306 Library Screened Results:
Table One: Top scoring ligand results from CH306 library with ligands docked into the active site of 4IL1 using GOLD.
Analysis: There were several high scoring ligands in this library that were docked into the homology model active site which is good and can be compared to other top scoring ligands in other libraries and the positive control ligands.
Excellent work -UM
Weeks 11 and 12
Trail Two of Protein Expression, Purification and Characterization
*gel is flipped
Figure One: SDS-PAGE Gel of T. brucei protein phosphatase 2B, putative after expression and purification. Lane two: Colorplus protein ladder. Lane three: sample zero containing pre-induction. Lane four: sample one containing protein after large culture overnight (lysate after induction). Lane five: sample two containing protein post-syringe filtration (soluble fraction). Lane six: sample three containing flow through. Lane seven: sample four containing wash. Lane eight: sample five containing first elution. Lane nine: sample six containing second elution.
Analysis: The same procedures were followed as the first trial and the same results were produced. In lane five - post syringe filtration it looks as if the protein has been lost. The next step will be to not syringe filter and add a higher concentration of NaCl at pH = 8 to the lysis buffer and try a sample with the higher concentration of NaCL but with 0.1% tritan and 5% glycerol to see if the protein shows up in a higher concentration after purification.
Trail Three Expression:
Figure Two: SDS-Page Gel of T. brucei protein phosphatase 2B, putative. Lane two contains the ColorPlus protein ladder. Lane three contains lysate after overnight induction. Lane four contains lysed supernatant with lysis buffer containing 100mM Tris, 500mM NaCl, and 10 mM Imidazole. Lane five contains lysed supernatant with lysis buffer containing 100mM Tris, 500mM NaCl, 10mM Imidazole, 5% glycerol and 01% Tritan. Lane six was supposed to contain nothing. Lane seven contains lysed supernatant with lysis buffer containing 100mM Tris, 500mM NaCl, and 10 mM Imidazole. Lane eight was supposed to contain nothing. Lane nine contains lysed supernatant with lysis buffer containing 100mM Tris, 500mM NaCl, 10mM Imidazole, 5% glycerol and 01% Tritan.
Analysis: This time instead of purifying the protein a gel was run on just the supernatant protein after expression spin down, lysis and spin down (no syringe filtration was done). Different lysis buffers containing a higher salt concentration and a higher salt concentration plus glycerol and tritan were used to make the protein more soluble, but what was determined is that the protein is not insoluble it is membrane bound. This means that instead of the protein being in the supernatant after the second spin down it is actually in the pellet containing the cell debris. Sadly I have to move to a different target now or try to work with the cell debris instead.
Virtual Progress:
Homology Model was made using SwissModel. The PDB number for the homology model is 4IL1.
Figure One: Crystal Structure of homology model 4IL1 from PDB.
Molprobity Results of 4IL1 after adding 77 Hydrogens with no flips.
Table 1: List of the nine positive control ligands found on PubChem and the five negative control ligands found on ZINC.
Figure Two: PyMOL image of 4IL1 shown as surface colored by elements carbons as green with FK5 ligand show as sticks extracted from template and moved to 4IL1.
Analysis: The homology model 4IL1 did not contain any ligands or a defined active site so a template model was aligned to the homology model and the ligand FK5 was extracted from the template and moved to the homology model. Then the active site was defined in this pocket using HERMES. The ligands were concatenated and after the homology model containing a defined active site was made a job was run and I am waiting for the results of how the ligands will score.
Weeks 9 and 10
Good analysis and data. Should include images from your virtual work. - BN
DNA Sequencing Results:
in the interest of saving you time grading my wikipage check my lab notebook for other clone results only the positive clone will be shown.
Figure One: BLAST of DNA sequencing sequence of clone #3 on master plate compared to DNA works sequence of T. brucei protein phosphatase 2B, putative. This is the forward read of the DNA sequencing sequence.
Figure Two: BLAST of DNA sequencing sequence of clone #3 on master plate compared to DNA works sequence of T. brucei protein phosphatase 2B, putative. This is the reverse read of the DNA sequencing sequence.
Analysis: Colony number #3 from round one of cloning is in fact a positive clone. The sequences BLASTed above are the sequences sent back to DNA sequencing from the first round. There is an N on both the forward and reverse spots, in different places in the sequence, but after looking at the chromotograph they are the correct base pairs so this clone is positive and matches T. brucei protein phosphatase 2B, putative. This clone will be used to move on to expression.
Protein Expression:
Round One of protein Expression Analysis:
Positive clone #3 was used to grow up in BL21(DE3) cells that was brought up to the correct concentration, harvested, lysed, spun down, and centrifuged. Samples 1 and 2 were collected throughout this process to be used for protein characterization, but first the final sample collected is purified.
Protein Purification:
Figure One: Nanodrop of T. brucei protein phosphatase 2B, putative protein from colony #3 after expression and purification (1st elution wash). Concentration of protein after first elution is 1.03 ng/uL.
Figure Two: Nanodrop of T. brucei protein phosphatase 2B, putative protein from colony #3 after expression and purification (2nd elution wash). Concentration of protein after second elution is 0.16 ng/uL.
Analysis: Protein sample from expression was purified with the help of a Ni-NTA resin attaching to the 6X his tag on the protein and only releasing when imidazole is present. So theoretically if it worked all of the protein should be in sample 5 ( first elution wash). According to the nanodrop the concentration of sample five is around 1.0 ng/uL. which can either be my protein or contamination. There was some concentration in sample six so not all of the protein was released after 1st elution or is once again contamination. Then I proceeded to characterization.
Protein Characterization
*gel is flipped
Figure One: SDS-PAGE Gel of T. brucei protein phosphatase 2B, putative after expression and purification. Lane two: Colorplus protein ladder. Lane three: sample one containing protein after large culture overnight (lysate after induction). Lane four: sample two containing protein pre-syringe filtration (soluble fraction). Lane five: sample three containing flow through. Lane six: sample four containing wash. Lane seven: sample five containing first elution. Lane eight: sample six containing second elution.
Analysis: After expression and purification the protein is not pure. Lane seven should show a distinct band at roughly 50 kDa, the size of my protein, but instead there is a lot of contamination. In lane three there is a distinct band were the protein is, but the protein was lost or the hist tag is buried deep within the protein so the Ni-NTA resin was not able to attach properly. Dimers do not seem to be the problems because they would occur at around 100 kDa and there is no band there in lane seven. Another problem could have been the buffers used during purification. So Anita and I made new buffers to see if that was the problem. Not enough imidazole might not have been in elution one so the protein was never released just the contamination. Next round of expression, purification, and characterization will be started with the new buffers.
Virtual:
- a homology model was made for T. brucei protein phosphatase 2B, putative and is 4IL1.
- also 9 positive control ligands and 5 negative controls ligands have been found using the binding database, pubchem, and ZINC.
Excellent work -UM
Weeks 7 and 8
PCR Squared:
Objective: The purpose of PCR squared is to make a lot of PCR because a lot will be lost during PCR clean up.
Figure One: PCR Squared of T. brucei protein phosphate 2B, putative on an agarose gel. Lane two contains the 100 bp ladder. Lanes three through six contains PCR squared solution. PCR squared solution contains diluted dNTP, secondary PCR from last trial, forward and reverse tail primers, reaction buffer, Q5 hotstart and autoclaved water.
Analysis: PCR Squared worked! A thick bright band appeared on the gel in the correct spot. PCR squared was made with the secondary PCR that worked (I used the 63 degC with full primary solution) and run again in the PCR machine this time with no gradient but at the temperature of 63 degC for the annealing temperature and all the other setting were kept the same as the secondary PCR. The purpose of PCR squared was to make extra PCR or PCR clean up purposes. Even though there was some contamination (most likely caused by the contamination in the secondary PCR solution) in the solutions it is still okay to move onto the next step, PCR cleanup. To determine the concentration of the target is good and can proceed to cloning.
PCR Cleanup:
Objective: The purpose of PCR clean up is to remove everything but the DNA from the PCR squared sample to get a good accurate concentration of the DNA.
Figure Two: Nanodrop of T. brucei protein phosphatase 2B, putative after cleaned up. Determined concentration to be 113.2 ng/uL.
Analysis: PCR cleanup gave good results of the DNA because the nanodrop found the DNA's concentration to be 113.2 ng/uL which is close to the actual concentration of T. brucei protein phosphatase 2B, putative. During the process of PCR clean up wash solution and elution buffer where added and centrifuged several times that way the DNA was able to separate completely from all the other solutions in the PCR squared solution. By the end only the DNA was left at the bottom of the tube and the concentration was then determined to be 113.2 ng/uL. This concentration is a little low but this might be because not all of the other substances were all filtered out which could have resulted in the DNA to be more diluted than it should be, but overall it still worked as it should have.
MidiPrep
Objective: The purpose of this lab is to extract pNIC-Bsa4 from perviously transformed bacterial cells.
Trial One:
Figure One: Nanodrop of pNIC-Bsa4 in DH5alpha from transformed bacterial cells after midiprep. Concentration is 91.6 ng/uL
Figure Two: Nanodrop of same pNIC-Bsa4 in DH5alpha from transformed bacterial cells after midiprep. Concentration is 88.9 ng/uL.
Trial Two:
Figure Three: Nanodrop of pNIC-Bsa4 in DH5alpha from different transformed bacterial cells after midiprep. Concentration is 55.3 ng/uL.
Figure Four: Nanodrop of same pNIC-Bsa4 in DH5alpha from different transformed bacterial cells after midiprep. Concentration is 54.5 ng/uL.
Analysis: In this experiment transformed pNIC-Bsa4 in DH5alpha was used to midiprep - a process that extracts the DNA of pNIC-Bsa4 from the transformed bacterial cells. From both rials a good concentration of the DNA in pNIC-Bsa4 was measure. The protocol states the A-260 10mm path value should be around one, and my values exceeded one which is good because pNIC-Bsa4 was not only successfully transformed but it was also successfully extracted from the transformed bacteria. In both trials a different plate of pNIC-Bsa4 was used. Trial two had to be done because after trial one sample was cut and PCR cleaned up the concentration of pNIC-Bsa4 left was extremely low and could not be used for cloning. But trial two will be used to clone!
Cloning T. brucei protein phosphatase 2B, putative in pNIC-Bsa4
Cutting Objective: The objective of cutting pNIC-Bsa4 is to prepare the DNA as an accepting vector to insert our target into.
Trial One:
Figure One: Nanodrop of pNIC-Bsa4 in DH5alpha after PCR cleanup in preparation to be an accepting vector. Concentration is 4.0 ng/uL.
Trial Two:
Figure Two: Nanodrop of new pNIC-Bsa4 in DH5alpha after PCR cleanup in preparation to be an accepting vector. Concentration is 49.9 ng/uL.
Analysis: In this experimen the solution made from midiprep was used. The pNIC-Bsa4 was cut using NEB Buffer 4, 100x BSA, and BsaI-HF in order to prepare pNIC-Bsa4 as an accepting vector that can accept T. brucei protein phosphatase 2B, putative for cloning. In trial one all of the sample (pNIC-Bsa4) from midiprep trial one was lost during PCR clean up or was cut wrong resulting in such a low concentration. If the concentration of pNIC-Bsa4 goes down a little after cutting and clean up, this is okay because the clean up is removing any contamination, but in trial one too much of the pNIC-Bsa4 was lost so we could not proceed to cloning. In trial two only about 5 ng/uL was lsot after cutting and clean up so this loss was contamination and the trial two of pNIC-Bsa4 (cut) is ready to proceed to cloning. Nothing was changed procedure wise during transformation, midiprep, and cutting for trial two only different plates of pNIC-Bsa4 was used and a new PCR clean up kit was used.
Cohesive End Generation, Transformation and Annealing Trial One:
Objective: The purpose of this lab is to prepare insert and accepting vector for successful cloning.
Figure One: T. brucei protein phosphatase 2B, putative with pNIC-Bsa4 on an LB+KAN (no suc) plate after overnight in the incubator.
Analysis: Although there were colonies on the plates both 1:2 and 1:3 ratios of vector to insert, LB+Kan+Suc plates were not used so pNIC-Bsa4 also grew with our DNA because sucrose kills pNIC. But there were some big colonies so putting a variety of colonies on a master plate with sucrose should show if our DNA grew.
Master Plate:
Objective: The purpose of making a master plate is to join insert and accepting vector together to later get positive matches from DNA sequencing.
Figure One: T. brucei protein phosphatase 2B, putative with accepting vector on a master plate with LB+Kan+Suc, grown overnight in the incubator.
Analysis: On the plate rows 1 and 3 is the 1:2 ratio alternating between big colony and little colony and on rows 2 and 4 the ratio 1:3 plates was used also alternating between big colony and little colony. It is good that the larger colonies grew on the plates because the smaller colonies from the first round of plates were supposed to be pNIC-Bsa4 because sucrose plates weren't used and the larger colonies were supposed to be our DNA. Because colonies grew the next step is to send the transformed tubes to DNA sequencing to check for matches. (When all eight tubes were spun down only the four that grew colonies formed a pellet because the other four didn't have any DNA).
Nanodrop check of Transformed Colonies:
Figure One: Nanodrop of T. brucei protein phosphate 2B, putative from colony #1 on master plate. Concentration of colony #1 is 154.6 ng/uL.
Figure Two: Nanodrop of T. brucei protein phosphate 2B, putative from colony #3 on master plate. Concentration of colony #3 is 157.2 ng/uL.
Figure Three: Nanodrop of T. brucei protein phosphate 2B, putative from colony #5 on master plate. Concentration of colony #5 is 159.3 ng/uL.
Figure Four: Nanodrop of T. brucei protein phosphate 2B, putative from colony #7 on master plate. Concentration of colony #7 is 161.2 ng/uL.
Then prepared these solutions and send to DNA sequencing. Waiting for Results
Gel Extraction:
Objective: The purpose of gel extraction is to reduce contamination of DNA by extracting from gel.
Figure One: Gel Check of T. brucei protein phosphatase 2B, putative of PCR squared (from secondary PCR at 63 degC for annealing temperature) to be used for gel extraction.
After gel extraction:
Figure Two: Nanodrop of half of DNA extracted from agarose gel. Concentration of DNA is 75.6 ng/uL.
Figure Three: Nanodrop of the other half of DNA extracted from agarose gel. Concentration of DNA is 73.3 ng/uL.
Analysis: After gel extraction (process where bands were cut from gel and purified using kit) the concentration of our DNA was determined using nanodrop to be around 74 ng/uL which is good value for the concentration to be proceeding into cloning. Because the graph of the DNA concentration of 73.3 ng/uL looks better this will be used as the insert for cloning trial two.
Cloning Trial Two:
Figure One: T. brucei protein phosphatase 2B, putative with accepting vector (pNIC-Bsa 4) on LB+ Kan+Suc plate after overnight incubation.
Analysis: Cloning round two did not work. The same accepting vector made two days prior was used and the new gel extracted DNA was used to make a new insert. The same steps were followed and this time was grown overnight on the correct plates with sucrose. The plates might not have grown because the plates could have been made wrong, the vector could have gone bad, the insert might not have worked properly or the ratios weren't correct. Now I wait for the results or proceed to next round of cloning with new plates, new insert and new vector with different ratios.
OMG this took forever to read... I'm glad after the 3rd trial it worked though :)! I'm loving the detailed analyses and figure captions. Keep up the groundbreaking research! - Michael T.
Weeks 5 and 6
Secondary PCR trials one through three done during week 5 :(
Objective: The purposed of secondary PCR is to take 1 uL of primary PCR that worked properly and PCR again with tail primers in order to run on a gel and get a thick band (with no contamination) the size of the target protein and later use for PCR squared.
Ladders used during primary and secondary PCR: 100 bp ladder and 1 kb ladder (100 bp ladder old) (100 bp ladder new) (1kb ladder)
Trial One:
(not sure why this image got distorted when placing on the wikipage)
Figure One: Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. In lane two the 1 kb ladder was used (we really should have used the 100 bp ladder), in lane three secondary PCR run with the primary PCR that was run for longer in the PCR machine. Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane seven contains 1 kb ladder and lane eight contains the secondary PCR with the primary PCR that was run for the shorter amount of time, also containing the same solutions.
Analysis:
Sadly secondary PCR did not work. A faint smear showed up in the wells kind of looking as if it was primary PCR but instead most likely contained contamination or the primers did not attach fully causing the DNA to not cut in the right spot. By using the 1 kb ladder we could not really tell if the protein was the correct size to begin with so next round we used the correct 100 bp ladder. Also we realized that we did not dilute the dNTP which could have also resulted in the secondary PCR to not work. Overall the time and the temperature needs to be changed that way the primers are able to latch onto DNA like they should.
Trial Two:
Figure Two: Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains a 100 bp ladder and lane three contains secondary PCR with the primary PCR that was run for longer in the PCR machine. Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane seven contains 100 bp ladder and lane eight contains the secondary PCR with the primary PCR that was run for the shorter amount of time, also containing the same solutions.
Analysis: Secondary PCR once again did not work. The main reason that the secondary PCR did not work was that once the samples were run in the PCR machine and we went to retrieve the samples the machine was not closed all the way during the entire run. This being said the samples were not properly run so the primers could not attach to the DNA and the DNA also did not replicate. The times and temperatures were not changed for the second trial of secondary PCR. Overall the time and the temperature needs to be changed and played with that way the primers are able to latch onto DNA like they should.
Trial Three (getting closer!)
Figure Three: Primary and Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains the 100 bp ladder, lane three contains primary PCR (new - containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart). Lane four contains secondary PCR run at a longer annealing time (30 seconds). Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane six contains 100 bp ladder, lane seven contains primary PCR and lane eight contains secondary PCR.
Analysis: Secondary PCR did not work again but getting on the right track! Primary PCR work perfectly, there is a bright smear starting where it should be and the primary PCR was run at the longer of the times given by the NEB guidelines. The secondary PCR on the other hand did not work again, but this time I changed the annealing time to 30 seconds that way the primers could attach themselves better the DNA/protein. There is a fant band where the size of the protein is but there is a smear in the lanes containing secondary PCR, this may have been caused by not all of the primers attaching. Next the temperature for annealing will be changed by either increasing or decreasing it a couple of degrees to see if the primers full attach themselves and thus produce a bright thick band the size of the protein.
Secondary PCR week six trials 4 - 9
Figure Four: Primary and Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains the 100 bp ladder, lane three contains primary PCR (new - containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart). Lane four contains secondary PCR run at a longer annealing time (30 seconds). Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane six contains 100 bp ladder, lane seven contains primary PCR and lane eight contains secondary PCR.
Analysis: Secondary PCR once again did not work. This time he annealing time was at 30 seconds and one solution was run at a lower annealing temperature of 55 degC and the other at a higher temperature of 62 degC. Nothing really showed up at the lower temperature but the high temperature shows some promise because there is a faint band. Secondary PCR might have worked if the primary turned out better because it was not even at the correct size because it should have been lower in the well. I was not happy with the way this gel turned out because it was pulled out and stopped several times so I ran another gel with Keely's and mine.
Figure Five: Primary and Secondary PCR of T. brucei protein phosphatase 2B, putative on an agarose gel. Lane two contains the 100 bp ladder, lane three contains a new primary PCR (containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart). Lane four contains an old PCR used in the first primary PCR. Lane five contains secondary PCR run at a longer annealing time (30 seconds) and a lower annealing temperature (55 degC). Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane six contains primary PCR and lane eight contains secondary PCR with the annealing time of 30 sec and annealing temperature of 62 degC. Lane seven contains Keely’s secondary PCR at the lower annealing temperature and lane eight contains Keely’s secondary PCR at the higher annealing temperature.
Analysis: Different gel with the same solutions made for previous gel. Once again the gel showed that both the old an new primary PCR did not come out as they should have causing the secondary to not work as it should have. There was once again a faint band with my secondary at the higher temperature but Keely's secondary's produced primer dimer (meaning primers didn't attach). The next step will be to remake primary again, ensure that it works first on a gel before making secondary then make secondary using a temperature gradient on the annealing temperature that is around 62 degC either higher or lower but mostly higher because the targets melting temperature is a lot higher than 58 degC.
Figure Six: Primary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains the 100 bp ladder. Lane three and four contains my primary PCR (containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart) with nothing changed with the NEB guidelines but all the times are at the highest. Lane five and six contain Keely’s primary PCR the same settings as mine.
Analysis: Primary PCR is no longer working like it should. It is too concentrated in one area of the smear which is causing a band to appear where the size of our DNA is. I did not change anything from the first time I ran primary PCR and it worked so this is slightly weird. The fact that the primary is too concentrated and bright might be caused by the oligo mix going bad. But we decided to run the secondary just incase because the primary is so concentrated it should have caused the secondary to work but it still did not. By the results of this primary an oligo mix needs to be remade that way the mix is fresh and will cause the primary and secondary to work.
Figure Seven: Secondary PCR of T. brucei protein phosphatase 2B, putative on an agarose gel. Lane two contains a 1 kb ladder (because 100 bp ladder was out). Lane three contains secondary PCR at the annealing temperature of 65 degC. Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane four contains secondary PCR at the annealing temperature of 64.5. Lane five contains secondary PCR run at the annealing temperature of 63 degC. Lane six contains secondary PCR run at the annealing temperature of 61.5 degC. All secondary PCR’s other times and temperatures were kept at the NEB guidelines.
Analysis: Yet again secondary PCR did not work. Lane six and lane five show some promise because a small band did appear but it is not as concentrated as it should be this might be caused by the fact that primary is so concentrated causing secondary to not be as concentrated as it needs to be. This secondary was made from the primary PCR shown in the previous gel so I did not expect secondary to work. Now I need to start completely over by making the oligo mix and seeing if this causes the primary to work and if I get a good primary then by moving onto secondary and trying different temperatures I should get a secondary PCR that works (hopefully).
Figure Eight: Primary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains 100 bp ladder. Lane three contains my primary PCR (with new oligo mix) containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart with the same settings as alsways. Lane four contains Keely’s primary PCR same as lane three. Lane four contains my primary PCR with 0.5 uL of oligo mix and 0.25 uL of Q5 hotstart. Lane five contains Keely’s primary PCR with half the oligo mix and half the Q5.
Analysis: Primary PCR was redone with a new oligo mix and still resulted in the primary PCR as too concentrated but not as bad as with the older oligo mix, the high concentration is spread throughout the smear more. But once the amount of oligo added was halved and the Q5 was halved my primary PCR produced better results than it has been producing. Keely’s primary PCRs are off though neither one of hers produced smears like mine did which is weird because we ran them on the same machine so she must have done something different with her actually PCR solution that produced these results. Both my primary PCR’s seem okay so the next step will be to take my primary PCR’s and run secondary using different annealing temperatures to see if secondary finally works.
Figure Nine: Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains a 100 bp ladder. Lane three contains secondary PCR with primary PCR halved at 62 degC annealing temperature. Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane four contains secondary PCR with normal primary PCR at 62 degC. Lane five contains secondary PCR with halved primary PCR at 63 degC. Lane six contains secondary PCR with normal primary PCR at 63 degC. Lane seven contains secondary PCR with halved primary PCR at 64 degC. Lane eight contains secondary PCR with normal primary PCR at 64 degC. Lane nine contains secondary PCR with halved primary PCR at 61 degC. Lane ten contains secondary PCR with normal primary PCR at 61 degC. All other times and temperatures remained the same.
Analysis: Secondary PCR finally worked (even though there is some contamination). All eight sample proved to be successful because finally a bring band appeared where our DNA size is; this is a new 100 bp ladder so it looks like it is the wrong size but its not because the ladder is lower. This time secondary PCR was made with the new primary with the new oligo mix. Halve of the secondary PCRs where made with the normal primary PCR that contained 1 uL of oligo and 0.5 uL of Q5 and the other half of the secondary PCRs contained the primary that had been halved (0.5 uL of oligo and 0.25 uL of Q5). Four were made from each set (either normal or halved primary) at the annealing temperatures of 61, 62, 63, or 64 degC and run at normal NEB guidelines for everything else. But it seems like the secondary PCR worked regardless of the normal or halved primary used, which means that the oligo mixed made at the beginning was not working properly. Now that we have lots of secondary that worked we can move on to PCR squared then curing African sleeping sickness.
*Also done on 10.4.13 and 10.5.13 we grew the overnight culture of pNIC-bsa4.
Week 3 and 4
Nicole - great work!. Dr. B 092713
ps - HF = high fidelity
PCR Trial Two and RE Digest (completed together)
Objective for PCR: The purpose of this experiment is to make a master mix also known as a coding sequence to make into a gel for analysis.
Objective for RE Digest: The purpose of this experiment was to digest pGBR22 plasmid (180.98 ng/uL) with restrictive enzymes EcoRI-HF and PvuII-HF and the buffer NEBuffer 4 and visualize the fragments on the agarose gel.
Figure One: Both RE Digest and PCR were run on this gel. Lanes 2-6 were RE Digest and Lanes 1, 7-10 were PCR. In this gel the RE Digest did not work as planned Lane 5's solution was not made correctly EcoRI-HF must have been added when PvuII-HF should have been added. Lane 2 contains 5 uL of 1kg DNA ladder, Lane 3 contains 12 uL of uncut plasmid (pGBR22 - 187.9 ng/uL), Lane 4 contains 30 uL of sample one with EcoRI-HF, Lane 5 contains 30 uL of sample two with PvuII-HF, and Lane 6 contains 30 uL of sample three with both EcoRI-HF and PvuII-HF (buffer used was NEBuffer 4). PCR did work as planned even though there was some contamination! Lane 1 contains 100bp DNA Ladder/marker, Lane 7 contains sample A with 0.0613 uL of plasmid (pGBR22 - 48.95 ng/uL), 5 uL of master mix, 1 uL of Taq and rest nanopure; Lane 8 contains sample B with .613 uL of plasmid, 5 uL mix, 1 uL Taq, rest nanopure; Lane 9 contains sample C with 6.13 uL plasmid, 5 uL mix, 1 uL Taq, rest nanopure; and Lane 10 contains no plasmid, 5 uL mix, 1 uL Taq, rest nanopure.
Figure Two: Both RE Digest and PCR were run on this gel. Lanes 1-6 were RE Digest and Lanes 7-10 were PCR. In this gel the RE Digest did work as planned. Lane one and two contain 5 uL of 1kg DNA ladder, Lane 3 contains 12 uL of uncut plasmid, Lane 4 contains 30 uL of sample one with EcoRI-HF, Lane 5 contains 30 uL of sample two with PvuII-HF, and Lane 6 contains 30 uL of sample three with both EcoRI-HF and PvuII-HF (buffer used was NEBuffer 4). PCR did work as planned even though there was some contamination! Lane 7 contains 100bp DNA Ladder/marker, Lane 8 contains sample A with 0.0613 uL of plasmid (pGBR22 - 108.98 ng/uL), 5 uL of master mix, 1 uL of Taq and rest nanopure; Lane 9 contains sample B with .613 uL of plasmid, 5 uL mix, 1 uL Taq, rest nanopure; Lane 10 contains sample C with 6.13 uL plasmid, 5 uL mix, 1 uL Taq, rest nanopure; and if there were a Lane 11 it would have contained no plasmid, 5 uL mix, 1 uL Taq, rest nanopure (but it was not put into the gel because we knew nothing would show up considering there was no plasmid in the sample).
Questions on RE Digest protocol:
The appropriate buffer to use for EcoRI-HF and PvuII-HF was NEBuffer 4. We used high frequency of the the two restrictive enzymes so we had to use that specific buffer, but it would have been a different buffer if did not use HF. The temperature recommend for incubation is between 65 degrees Celsius and 80 degrees Celsius. The conditions to stop EcoRI-HF buffer is 65 degrees Celsius for 20 minutes and the conditions to stop PuvII-HF buffer is 80 degrees Celsius for 20 minutes.
Analysis for PCR:
The experiment was done over again with two different trials. The only difference between trial one and trial two was the anneal temperature. In trial one the anneal temperature was set to 42 degC while trial two stayed at the 45 degC listed on the protocol. We did this to see if it would produce different outcomes, but it did not that we are aware of because both of the trails worked. PCR worked this time because the master mix was made correctly, everything was kept at the correct temperature, Taq was added last and the samples were immediately put into the PCR machine unlike last time where the Taq was left out for a while before being put into the solutions. There was some contamination that showed up in the gel, but the thicker lines are at the correct size according the the 100 kb ladder.
Analysis for RE Digest:
In this experiment six samples were made two with EcoRI-HF, two with PvuII-HF and two with both along with the buffer NEBuffer 4 and nanopure. These samples were incubated at 37 degC for 2 hours, spun down, put on a heat block for 20 minutes between the temperatures of 65 degC and 80 degC and spun down again. After this a gel was made with a 1kb DNA ladder, uncut plasmid (but a different plasmid was used not the same as the plasmid used to make the samples because the plasmid was used up; uncut plasmid used was pGBR22 187.9 ng/ul) and the three samples (all done on two different gels). The trial one gel did not work because one of the samples was made incorrectly, I believe I accidentally put the same restrictive enzyme (EcoRI-HF) in both samples one and two. But the trial two experiment was done perfectly and the sizes are correct for each of the samples.
Future:
Both of the experiment are extremely important in the future when both of the processes will have to repeated again with our target. RE Digest is especially important when we will want to cut the plasmid and find what can bind/fit into those cuts.
Primer Overlap (with Q5 Polymerase)
Objective: Thaw out primer to then mix each cell by pipetting up and down then remove 1 uL of plasmid from each cell to add with autoclaved water, then freeze for further research. .
Analysis:
In this experiment a primer was made using 1 uL of primer from each cell. Since T. brucei protein phosphatase 2B, putative had 36 cells 74 uL of autoclaved water was added to the tube to give the solution a total volume of 100 uL. Once completed the sample was put into the -20 degC freezer. This experiment was not been completed but will be in the future.
PCR Primer Design Tails for pNIC-Bsa4 Cloning
Objective: The purpose of this lab was to design the forward and reverse primer of T. brucei protein phosphatase 2B, putative and insert in pNIC-Bsa4.
Coding Sequence for T. Brucei protein phosphatase 2B, putative TACTTCCAATCCATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGATGGTCTCAAAGAACGTGAATACGTTTGGAAAAAATCTCACGCGGACGAGTCCCCGG GTTCTAACCCGCAAATCCTGCCGACCCTGGTTCTGCCGCACCACCTCGTGTTCGACAACGACGGTGCGCCACTGGCGGACAACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCTCCACGTTGAGGATGCGCTGAATATCGTTCACCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCTGCAGTAGTCTGCGGTGATCTCC ACGGCCAGTTCCACGACCTCCTGACCCTGCTGGAAGTTAATGGTCACCCTAGCGTTCAACAGTACGTTTTCCTGGGTGACTACGTTGACCGTGGTGACTTCTCTGCTGAAA TCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCCGCGCTCTTTCATCCTGCTGCGTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAGGAAAT CGAATCTAAATACTCTTCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTGTCTTGCGTTGTTAACGACCGTTTCTTTTGTGTTCATGGTGGTCTCTCTCC GCTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGTGAAACCCCGTCTACTGGTCCGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACC GACTGTGCGACGCCGAGCGAAGAACTGTTCGTTTTTAACACTAAACGTGGTTGCTCTTACAACTACAGCTACGAAGCCGTTTGCCGTTTCCTCGAAGCGAACAATCTGTGCA CGGTTATTCGTGGTCATGAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTGTTCCGGCGGTTGTATGTGTTTTTAGCGCGAGCAATTATTGCGGTAC CTACGGTAACATGGCCGCAGTTGTAGCGATCGATGGTGACGTTATGAACATCCGTCAGTACATGGCGACCTCTCACGACTCTTGCACCCCTAACCACTTCAATGCGATCTC TCGTATGCAGCCTCTGGCGATCCATGAGGCGGTAGAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAAAAAGTTGAGGTTGAAAAAAACCT GTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGCGATATGATCTGCGCAATGCACCACATTGTTACCCAGTAAAGGTGGATA
Forward Primer (33 base pairs) TACTTCCAATCCATGTCTTCTGGCGCGTCCCAT (order) Melting Temperature 0 mM Mg 66.8 degC 1.5 mM Mg 73.6 degC 2 mM Mg 74.1 degC 4 mM Mg 75.0 degC 6 mM Mg 75.4 degC Reverse Primer (38 base pairs) GCGCAATGCACCACATTGTTACCCAGTAAAGGTGGATA Reverse complement TATCCACCTTTACTGGGTAACAATGTGGTGCATTGCGC (order) Melting Temperature 0 mM Mg 66.4 degC 1.5 mM Mg 73.7 degC 2 mM Mg 74.1 degC 4 mM Mg 74.9 degC 6 mM Mg 75.3 degC
Figure One: Virtual gel of CDS sequence of T. brucei protein phosphatase 2B, putative with enzyme BsaI.
Figure Two: Virtual gel of pNIC28-Bsa4 sequence with enzyme BsaI.
Figure Three: Virtual gel of CDS of T. brucei protein phosphatase 2B, putative in pNIC28-Bsa4 with enzyme BsaI.
Analysis:
In this experiment the coding sequence for T. brucei protein phosphatase 2B, putative was used along with upstream and downstream primers for LIC cloning to make forward and reverse primers to be ordered for later experimentation. The coding sequence was added to pNIC-Bsa4 to create a final sequence with no SacB gene. In order to get the melting temperatures the same between the forward and reverse primers more amino acids (bases) were added to the reverse that way when they were both at 2mM Mg (and other standards) that the melting temperatures were both 74.1 degC. 33 base pairs for forward primer and 38 base pairs for reverse primer were needed to have the same melting temperatures. The reverse complement will be ordered later because the primer will read oppositely which is why the reverse must be ordered. After ordering the next step will be to see what can fit into the SacB gene of out target's plasmid.
Primary PCR
Objective: The purpose of this lab was to make the primary PCR solution with our target's diluted template/primer to see if it showed up on an agarose gel.
Figure One: Primary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two has the 1kg DNA ladder and lane three has the primary PCR containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart. Lane three is different than lane eight because it was run in the PCR machine for the longer of the NEB recommended guidelines for time while lane eight had the shorter cycle times. Lane seven contains 1kg DNA ladder and lane eight contains the primary PCR solution at the shorter cycle time.
Figure Two: 1kg Ladder used in gel above.
Analysis:
In this experiment a primary PCR solution was made using 5X rxn buffer, diluted dNTP, our oligo mix, dH2O and Q5 hotstart polymerase and run through the PCR machine with the NEB recommend guidelines. One solution was run at the higher ends of cycle time and the other solution was run at the lower end of the cycle times listed. The solutions remained in the PCR machine overnight then were moved to the -20 degC freeze before running the gel. The gel showed that the primary PCR did work because there is a smear in that well where the solution was put in; in lane three the smear was around 1.8 kb which is the size of our target. The primary PCR that was run for the longer time did show up better than the one that ran for a shorter time, but both worked. The next step is the do the secondary PCR with the forward and reverse primers ordered earlier this week, but we are waiting for the primers to come in in order to start the next step.
Week 1 and 2
Nicole, great job. Crop your agarose gel images (can do in the software) to remove alot of the black. Re-do PCR this week.. Dr. B 090913
Nanodrop Spectorphotometer: Determine Concentration of Plasmids, DNA
Objective:
The purpose of this experiment was to relearn how to use the nanodrop spectrophotometer for future experiments and to properly take measurements of pGBR22.
Images:
Figure One: Plot of pGBR22 (205.4 ng/uL) using the nanodrop spectrophotometer that found the concentration to be 202.8 ng/uL.
Figure Two: Plot of pGBR22 (205.4 ng/uL) using the nanodrop spectrophotometer that found the concentration to be 206.8 ng/uL.
Results/Conclusion:
This experiment refreshed and reinforced how to properly take measurements utilizing the nanodrop spectrophotometer. pGBR22's original concentration is 205.4 ng/uL. The first measurement found the concentration to be 202.8 ng/uL with a 260/280 value of 1.98 and a 260/230 value to be 1.83, which is close to the original concentration value. The second measurement found the concentration to be 206.8 ng/uL with a 260/280 value of1.94 and a 260/230 value to be 1.81. The second concentration value was also close to the original concentration value therefore both measurements were valid. The average of the two concentrations measured was found to be 204.8 ng/uL. Errors could have been made during the experiment if the machine was not properly blanked skewing future data or some of the water may not have been wiped off before the first measurement was taken causing the concentration to dilute a small amount possibly accounting for the reason that the first measurements concentration was lower than the second.
Nanodrop Spectrophotometer: Determine Concentration of Plasmid
Objective:
The purpose of this lab was to prepare a plasmid for the core lab for them to add a primer to the solution for later analysis.
In this experiment option A was chosen.
Images:
Figure One: Documented proof that a request was sent using core website requesting for in-house primer M13F to be add and DNA sequencing to be done on the prepared template of DNA (pGBR22).
Figure Two: Results of the DNA sequencing on the DNA template + primer (M13F) done at the core lab.
Figure Three: Nucleotide BLAST of the coding sequence against Human and added all nucleotide collection databases.
Results/Conclusion:
In this experiment a DNA template using pGBR22 was made and taken to a different lab after the proper request form was sent in. The primer chosen was M13F and the template was taken to the lab the next day after making. Results were emailed several hours later, the analysis of these results will take place in the Analyzing DNA Sequence lab. Since option A was chosen for the experiment several errors could have occurred at the other lab that we have no control over such as using the wrong primer, not adding the correct amount, maybe the primer was not kept at the right temperature, this error also could have occurred in our lab as well if the pGBR22 was not kept at the right temperature during preparation.
PCR Primer Design for Primer Overlap Assembly PCR:
Objective:
The purpose of this experiment was to design an oligo set of primers to be order for further experimentation involving cloning.
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| Helix Systems -- Center for Information Technology |
| http://helix.nih.gov |
| National Institutes of Health, Department of Health and Human Services |
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| DNAWorks Web Site: http://helixweb.nih.gov/dnaworks |
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| Send all correspondence to webtools@helix.nih.gov |
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Job started on 09/03/2013 at 14:40:38
Job name: NW9022013TBP2B
Output will be sent to nicole_welch23@yahoo.com
SEQUENCE 1: PROTEIN LENGTH = 431
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1 MSSGASHHELTRGRDGLKEREYVWKKSHADESPGSNPQILPTLVLPHHLVFDNDGAPLAD
61 NIKVHFGRGWRLHVEDALNIVHRCALIMKEEPNVVRLKGSAVVCGDLHGQFHDLLTLLEV
121 NGHPSVQQYVFLGDYVDRGDFSAEIVLLCMSFKLLYPRSFILLRGNHESRQLTSCFNFKQ
181 EIESKYSSMVYEEIMAAFDCFPLSCVVNDRFFCVHGGLSPLLTYLGEIDTVNRFRETPST
241 GPMCDLLWSDPMFGDDTDCATPSEELFVFNTKRGCSYNYSYEAVCRFLEANNLCTVIRGH
301 ETQPGGYKLYRHTPKGVPAVVCVFSASNYCGTYGNMAAVVAIDGDVMNIRQYMATSHDSC
361 TPNHFNAISRMQPLAIHEAVEKWCVASHGGSSGDAKAEKVEVEKNLSEDDSSVVLREKLG
421 DMICAMHHIVT
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None found
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32 oligonucleotides need to be synthesized
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1 ATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGAT 45
2 CCGCGTGAGATTTTTTCCAAACGTATTCACGTTCTTTGAGACCATCACGACCACGGGTCA 60
3 TTTGGAAAAAATCTCACGCGGACGAGTCCCCGGGTTCTAACCCGCAAATCCTGCCGACCC 60
4 GGCGCACCGTCGTTGTCGAACACGAGGTGGTGCGGCAGAACCAGGGTCGGCAGGATTTGC 60
5 CAACGACGGTGCGCCACTGGCGGACAACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCT 60
6 GATCAGCGCGCAACGGTGAACGATATTCAGCGCATCCTCAACGTGGAGGCGCCAACCGCG 60
7 CCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCTGCAGT 60
8 GTCAGGAGGTCGTGGAACTGGCCGTGGAGATCACCGCAGACTACTGCAGAGCCCTTCAGG 60
9 AGTTCCACGACCTCCTGACCCTGCTGGAAGTTAATGGTCACCCTAGCGTTCAACAGTACG 60
10 GCAGAGAAGTCACCACGGTCAACGTAGTCACCCAGGAAAACGTACTGTTGAACGCTAGGG 60
11 CCGTGGTGACTTCTCTGCTGAAATCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCC 60
12 CGAGACTCGTGGTTACCACGCAGCAGGATGAAAGAGCGCGGGTACAGCAGTTTGAAAGAC 60
13 GTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAGGAAATCGAAT 60
14 CCATGATTTCTTCGTAAACCATAGAAGAGTATTTAGATTCGATTTCCTGTTTGAAGTTGA 60
15 TCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTGTCTTGCGTTGTT 60
16 CAGCGGAGAGAGACCACCATGAACACAAAAGAAACGGTCGTTAACAACGCAAGACAGCGG 60
17 GGTGGTCTCTCTCCGCTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGT 60
18 CAGAGCAGGTCACACATCGGACCAGTAGACGGGGTTTCACGAAAACGGTTAACAGTATCG 60
19 CGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACCGACTGTGCGACGC 60
20 AGAGCAACCACGTTTAGTGTTAAAAACGAACAGTTCTTCGCTCGGCGTCGCACAGTCGGT 60
21 AACACTAAACGTGGTTGCTCTTACAACTACAGCTACGAAGCCGTTTGCCGTTTCCTCGAA 60
22 TGGGTCTCATGACCACGAATAACCGTGCACAGATTGTTCGCTTCGAGGAAACGGCAAACG 60
23 TTCGTGGTCATGAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTG 60
24 CGCAATAATTGCTCGCGCTAAAAACACATACAACCGCCGGAACACCCTTTGGGGTATGGC 60
25 GCGCGAGCAATTATTGCGGTACCTACGGTAACATGGCCGCAGTTGTAGCGATCGATGGTG 60
26 TCGTGAGAGGTCGCCATGTACTGACGGATGTTCATAACGTCACCATCGATCGCTACAACT 60
27 CATGGCGACCTCTCACGACTCTTGCACCCCTAACCACTTCAATGCGATCTCTCGTATGCA 60
28 ACACACCACTTTTCTACCGCCTCATGGATCGCCAGAGGCTGCATACGAGAGATCGCATTG 60
29 GCGGTAGAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAA 60
30 CAGAGCTGTCATCTTCAGACAGGTTTTTTTCAACCTCAACTTTTTCCGCTTTCGCGTCAC 60
31 TGTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGCGATATGATCTGCGCAA 60
32 GGTAACAATGTGGTGCATTGCGCAGATCATATCGCC 36
FINAL SUMMARY FOR 1 SOLUTION
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# Tm Len | Score TmRange Short Long #Olig #Repeat #Misprime
1 62 60 | 0.000 1.9 14 60 32 0 0
Figure One: Final results logfile of T. brucei protein phosphatase 2B, putative using the amino acid sequence found on the TDR website.
Protein Length for T. brucei protein phosphatase 2B, putative = 431
Results/Conclusion:
In this experiment the amino acid sequence for T. brucei protein phosphatase 2B, putative was used to find oligoneleotides (primers) to be order for further experiments regarding this target. An error occurred during this experiment because a wrong YP number was given on the Wiki for this target so the experiment was redone with the correct amino acid sequence. Once this problem was fixed the correct oligos were found and are ready to be ordered.
PCR
Objective: The purpose of this experiment is to make a master mix also known as a coding sequence to make into a gel for analysis.
Figure One: Agarose gel with 1kb DNA ladder showing in the second lane. In lane three sample A a 1:1000 dilution template, lane four sample B a 1:1000 dilution differing in the amount of DDW, lane give sample C a 1:100 dilution and lane six sample D with no DNA (control sample). Only the ladder is visible. The picture was taken in the UV imaging box.
Analysis/Conclusions:
In this experiment sample A-D were made differing in the amount of DNA template, template dilution and diluted Taq, and were run in the PCR machine. Once finished in the machine the agarose gel was made but proved to be unsuccessful because only the ladder showed up in the image, and at least one of the samples should have shown up. Several errors could have been made such as the right amounts were not added into each sample, the dilutions were made incorrectly, certain solutions and the samples were not kept at the correct temperature or the gel did not work by expressing the DNA like it should have.
Analyzing DNA Sequencing
Objective: The purpose of this lab was to BLAST sequences and compare sequences in order to determine the DNA sequence of a plasmid.
Figure One: Screenshot of Analyzing DNA Sequencing that is one the Google Drive.
Results/Conclusions:
In this experiment a primer sequence was used and altered in order to make a final reverse complement sequence with part of the original sequence in it. The final sequence was put into NEB where the primer was placed in the primer ring where a gel could be found.
Enzyme Assay of yopH surrogate protein:
Figure One: Enzyme assay of yopH at different enzyme concentration amounts using a spectrophotometer read at 410 nm.
Figure Two: Graph of average absorbance read at 410 nm versus enzyme (yopH) concentration for the first trial.
Analysis: yopH surragote protein was used to run an enzyme assay and inhibition assay after it was properly expressed and purified. As more yopH was added to the solution more activity was oberserved because the solution turned more and more yellow after the addition of NaOH and there was an increase in absorbance measured for each solution with more yopH concentration. This being said the enzyme assay worked and the 10uL amount of yopH enzyme dilution will be used for inhibition assay because there is a significant jump from 5 uL to 10 uL and should properly be inhibited at this protein amount.
Inhibition Assay of yopH using 5852635 and 5250098:
Figure one: Inhibition assay of yopH against 5252635 inhibitor compound at different concentrations of the compound using a spectrophotometer read at 410 nm.
Figure Two: Graph of inhibition assay trial one using the inhibitory compound 5852635 against yopH. Absorbance at 410 nm versus inhibitor compound concentration.
Analysis: An inhibition assay was run with the yopH surrogate protein against a protein phosphatase inhibitor 5852635. When there was no concentration of inhibitor in the sample the absorbance reading was very similar to the absorbance of yopH at the 10 uL amount form the enzyme assay so this shows consistency which is good. Then once the inhibitor was addd to the solutions the activity of yopH greatly decreased. At the concentration of 0.6 for the inhibitor compound there was a slight increase in absorbance this could have been because at that high of a inhibitor concentration it is not optimal. The ortho positive control inhibitor is a good comparing point for the amount of activity that the protein should have in the presence of an inhibitor and because the activity is around the positive inhibitor absorbance this is really good! There was not a steady decrease like what was to be expected but this could be because at the inhibitor concentration at 0.1 it already inhibits the protein very well. This being said the next trial should be tested using even lower concentration of the inhibitor compound to see if this results in a more steady decrease.
Figure Three: Inhibition assay of yopH against 5250098 inhibitor compound at different inhibitor concentrations using a spectrophotometer read at 410 nm.
Figure Four: Graph of inhibition assay trial one using the inhibitory compound 5250098 against yopH. Absorbance at 410 nm versus inhibitor concentration.
Analysis: The same procedures were followed for this inhibition assay as the one that worked with the protein amount still at 10 uL, this timing using a different inhibitory compound ( 5250098). This compound did not inhibit the activity of yopH compared to the positive inhibitor. The samples where not inhibitor was added were consistent with the absorbance from the enzyme assay at 10 uL which is good, but the compound did not work to inhibit yopH activity. This is because the activity (read by absorbance) of the enzyme was not decreased. This could have been operator error or equipment error but both inhibition trials were run at the same time so that might not have been the case. Therefore this compound is not going to be used for further testing.
Virtual:
NIH library-
Table One: Top scoring ligand results from NIH library with the ligands docked into the active site of 4IL1 using GOLD.
Analysis: The NIH library, containing 446 ligands, was screened using GOLD and the top 46 scoring ligands are shown in the table above. There were two ligands that scored in the 90's which is really good and those two could potentially be good inhibitors of T. brucei protein phosphatase 2B. Also all 46 highest scoring ligands has really high scores as well which is good! The next step will be to compare these results to ICM results to see if there might be a good inhibitor.
HF9PlatesPlates Library-
Table Two: Top scoring ligand results form HF9PlatesPlates library with the ligands docked into the active site of 4IL1 using GOLD.
Analysis: The HF9PlatesPlates library, containing 400 ligands, was screened using GOLD and the top 40 scoring ligands are shown in the table above. These scores are not as good as the scores from the NIH library but the results are better than the CH306 library results. The few highest scoring ligands may or may not be good inhibitors but the next step will be to run this library through ICM and compare the results to see if there is a potentially good inhibitor.
Weeks 13 and 14
*week 13: tried expressing surrogate plates and found that the yopH plate was too old to use so I could not grow anything and the ftHAP plate was not expressing properly so new yopH plates were made and used for expression, purification and characterization.
Week 14:
Figure One: SDS-PAGE Gel of yopH surrogate protein after expression and purification. Lane two: Colorplus protein ladder. Lane three: sample one containing protein after large culture overnight (lysate after induction). Lane four: sample two containing protein pre-syringe filtration (soluble fraction). Lane five: sample three containing flow through. Lane six: sample four containing wash. Lane seven: sample five containing first elution. Lane eight: sample six containing second elution.
Analysis: Expression, purification and characterization finally worked for yopH!! Option A was followed with the normal procedures for expression and purification, and the normal lysis buffer was used. Elution one does not have much contamination so FPLC can be optional. May try FPLC with only have of the elution one solution that way the protein is not lost during FPLC and there will still be some to do enzyme assays with.
Virtual on my target: T. brucei protein phosphatase 2B
Table One: Results of positive and negative controls ligands docked into the active site of the homology model of 4IL1.
Analysis: The top three highest scores were 141.88, 129.95, and 121.29 all of which are positive control ligands which is a good thing because a high score for a negative control ligand would mean something is wrong with the defined active site. Because there were three positive ligands with high scores now 4IL1 can be screened against several libraries and compare the results with the three high scoring positive ligands to determine if any novel ligands can be found that would inhibit T. brucei protein phosphatase 2B, even though enzyme assays and inhibition assays will not be run with this protein.
CH306 Library Screened Results:
Table One: Top scoring ligand results from CH306 library with ligands docked into the active site of 4IL1 using GOLD.
Analysis: There were several high scoring ligands in this library that were docked into the homology model active site which is good and can be compared to other top scoring ligands in other libraries and the positive control ligands.
Excellent work -UM
Weeks 11 and 12
Trail Two of Protein Expression, Purification and Characterization
*gel is flipped
Figure One: SDS-PAGE Gel of T. brucei protein phosphatase 2B, putative after expression and purification. Lane two: Colorplus protein ladder. Lane three: sample zero containing pre-induction. Lane four: sample one containing protein after large culture overnight (lysate after induction). Lane five: sample two containing protein post-syringe filtration (soluble fraction). Lane six: sample three containing flow through. Lane seven: sample four containing wash. Lane eight: sample five containing first elution. Lane nine: sample six containing second elution.
Analysis: The same procedures were followed as the first trial and the same results were produced. In lane five - post syringe filtration it looks as if the protein has been lost. The next step will be to not syringe filter and add a higher concentration of NaCl at pH = 8 to the lysis buffer and try a sample with the higher concentration of NaCL but with 0.1% tritan and 5% glycerol to see if the protein shows up in a higher concentration after purification.
Trail Three Expression:
Figure Two: SDS-Page Gel of T. brucei protein phosphatase 2B, putative. Lane two contains the ColorPlus protein ladder. Lane three contains lysate after overnight induction. Lane four contains lysed supernatant with lysis buffer containing 100mM Tris, 500mM NaCl, and 10 mM Imidazole. Lane five contains lysed supernatant with lysis buffer containing 100mM Tris, 500mM NaCl, 10mM Imidazole, 5% glycerol and 01% Tritan. Lane six was supposed to contain nothing. Lane seven contains lysed supernatant with lysis buffer containing 100mM Tris, 500mM NaCl, and 10 mM Imidazole. Lane eight was supposed to contain nothing. Lane nine contains lysed supernatant with lysis buffer containing 100mM Tris, 500mM NaCl, 10mM Imidazole, 5% glycerol and 01% Tritan.
Analysis: This time instead of purifying the protein a gel was run on just the supernatant protein after expression spin down, lysis and spin down (no syringe filtration was done). Different lysis buffers containing a higher salt concentration and a higher salt concentration plus glycerol and tritan were used to make the protein more soluble, but what was determined is that the protein is not insoluble it is membrane bound. This means that instead of the protein being in the supernatant after the second spin down it is actually in the pellet containing the cell debris. Sadly I have to move to a different target now or try to work with the cell debris instead.
Virtual Progress:
Homology Model was made using SwissModel. The PDB number for the homology model is 4IL1.
Figure One: Crystal Structure of homology model 4IL1 from PDB.
Table 1: List of the nine positive control ligands found on PubChem and the five negative control ligands found on ZINC.
Figure Two: PyMOL image of 4IL1 shown as surface colored by elements carbons as green with FK5 ligand show as sticks extracted from template and moved to 4IL1.
Analysis: The homology model 4IL1 did not contain any ligands or a defined active site so a template model was aligned to the homology model and the ligand FK5 was extracted from the template and moved to the homology model. Then the active site was defined in this pocket using HERMES. The ligands were concatenated and after the homology model containing a defined active site was made a job was run and I am waiting for the results of how the ligands will score.
Weeks 9 and 10
Good analysis and data. Should include images from your virtual work. - BN
DNA Sequencing Results:
in the interest of saving you time grading my wikipage check my lab notebook for other clone results only the positive clone will be shown.
Figure One: BLAST of DNA sequencing sequence of clone #3 on master plate compared to DNA works sequence of T. brucei protein phosphatase 2B, putative. This is the forward read of the DNA sequencing sequence.
Figure Two: BLAST of DNA sequencing sequence of clone #3 on master plate compared to DNA works sequence of T. brucei protein phosphatase 2B, putative. This is the reverse read of the DNA sequencing sequence.
Analysis: Colony number #3 from round one of cloning is in fact a positive clone. The sequences BLASTed above are the sequences sent back to DNA sequencing from the first round. There is an N on both the forward and reverse spots, in different places in the sequence, but after looking at the chromotograph they are the correct base pairs so this clone is positive and matches T. brucei protein phosphatase 2B, putative. This clone will be used to move on to expression.
Protein Expression:
Round One of protein Expression Analysis:
Positive clone #3 was used to grow up in BL21(DE3) cells that was brought up to the correct concentration, harvested, lysed, spun down, and centrifuged. Samples 1 and 2 were collected throughout this process to be used for protein characterization, but first the final sample collected is purified.
Protein Purification:
Figure One: Nanodrop of T. brucei protein phosphatase 2B, putative protein from colony #3 after expression and purification (1st elution wash). Concentration of protein after first elution is 1.03 ng/uL.
Figure Two: Nanodrop of T. brucei protein phosphatase 2B, putative protein from colony #3 after expression and purification (2nd elution wash). Concentration of protein after second elution is 0.16 ng/uL.
Analysis: Protein sample from expression was purified with the help of a Ni-NTA resin attaching to the 6X his tag on the protein and only releasing when imidazole is present. So theoretically if it worked all of the protein should be in sample 5 ( first elution wash). According to the nanodrop the concentration of sample five is around 1.0 ng/uL. which can either be my protein or contamination. There was some concentration in sample six so not all of the protein was released after 1st elution or is once again contamination. Then I proceeded to characterization.
Protein Characterization
*gel is flipped
Figure One: SDS-PAGE Gel of T. brucei protein phosphatase 2B, putative after expression and purification. Lane two: Colorplus protein ladder. Lane three: sample one containing protein after large culture overnight (lysate after induction). Lane four: sample two containing protein pre-syringe filtration (soluble fraction). Lane five: sample three containing flow through. Lane six: sample four containing wash. Lane seven: sample five containing first elution. Lane eight: sample six containing second elution.
Analysis: After expression and purification the protein is not pure. Lane seven should show a distinct band at roughly 50 kDa, the size of my protein, but instead there is a lot of contamination. In lane three there is a distinct band were the protein is, but the protein was lost or the hist tag is buried deep within the protein so the Ni-NTA resin was not able to attach properly. Dimers do not seem to be the problems because they would occur at around 100 kDa and there is no band there in lane seven. Another problem could have been the buffers used during purification. So Anita and I made new buffers to see if that was the problem. Not enough imidazole might not have been in elution one so the protein was never released just the contamination. Next round of expression, purification, and characterization will be started with the new buffers.
Virtual:
- a homology model was made for T. brucei protein phosphatase 2B, putative and is 4IL1.
- also 9 positive control ligands and 5 negative controls ligands have been found using the binding database, pubchem, and ZINC.
Excellent work -UM
Weeks 7 and 8
PCR Squared:
Objective: The purpose of PCR squared is to make a lot of PCR because a lot will be lost during PCR clean up.
Figure One: PCR Squared of T. brucei protein phosphate 2B, putative on an agarose gel. Lane two contains the 100 bp ladder. Lanes three through six contains PCR squared solution. PCR squared solution contains diluted dNTP, secondary PCR from last trial, forward and reverse tail primers, reaction buffer, Q5 hotstart and autoclaved water.
Analysis: PCR Squared worked! A thick bright band appeared on the gel in the correct spot. PCR squared was made with the secondary PCR that worked (I used the 63 degC with full primary solution) and run again in the PCR machine this time with no gradient but at the temperature of 63 degC for the annealing temperature and all the other setting were kept the same as the secondary PCR. The purpose of PCR squared was to make extra PCR or PCR clean up purposes. Even though there was some contamination (most likely caused by the contamination in the secondary PCR solution) in the solutions it is still okay to move onto the next step, PCR cleanup. To determine the concentration of the target is good and can proceed to cloning.
PCR Cleanup:
Objective: The purpose of PCR clean up is to remove everything but the DNA from the PCR squared sample to get a good accurate concentration of the DNA.
Figure Two: Nanodrop of T. brucei protein phosphatase 2B, putative after cleaned up. Determined concentration to be 113.2 ng/uL.
Analysis: PCR cleanup gave good results of the DNA because the nanodrop found the DNA's concentration to be 113.2 ng/uL which is close to the actual concentration of T. brucei protein phosphatase 2B, putative. During the process of PCR clean up wash solution and elution buffer where added and centrifuged several times that way the DNA was able to separate completely from all the other solutions in the PCR squared solution. By the end only the DNA was left at the bottom of the tube and the concentration was then determined to be 113.2 ng/uL. This concentration is a little low but this might be because not all of the other substances were all filtered out which could have resulted in the DNA to be more diluted than it should be, but overall it still worked as it should have.
MidiPrep
Objective: The purpose of this lab is to extract pNIC-Bsa4 from perviously transformed bacterial cells.
Trial One:
Figure One: Nanodrop of pNIC-Bsa4 in DH5alpha from transformed bacterial cells after midiprep. Concentration is 91.6 ng/uL
Figure Two: Nanodrop of same pNIC-Bsa4 in DH5alpha from transformed bacterial cells after midiprep. Concentration is 88.9 ng/uL.
Trial Two:
Figure Three: Nanodrop of pNIC-Bsa4 in DH5alpha from different transformed bacterial cells after midiprep. Concentration is 55.3 ng/uL.
Figure Four: Nanodrop of same pNIC-Bsa4 in DH5alpha from different transformed bacterial cells after midiprep. Concentration is 54.5 ng/uL.
Analysis: In this experiment transformed pNIC-Bsa4 in DH5alpha was used to midiprep - a process that extracts the DNA of pNIC-Bsa4 from the transformed bacterial cells. From both rials a good concentration of the DNA in pNIC-Bsa4 was measure. The protocol states the A-260 10mm path value should be around one, and my values exceeded one which is good because pNIC-Bsa4 was not only successfully transformed but it was also successfully extracted from the transformed bacteria. In both trials a different plate of pNIC-Bsa4 was used. Trial two had to be done because after trial one sample was cut and PCR cleaned up the concentration of pNIC-Bsa4 left was extremely low and could not be used for cloning. But trial two will be used to clone!
Cloning T. brucei protein phosphatase 2B, putative in pNIC-Bsa4
Cutting
Objective: The objective of cutting pNIC-Bsa4 is to prepare the DNA as an accepting vector to insert our target into.
Trial One:
Figure One: Nanodrop of pNIC-Bsa4 in DH5alpha after PCR cleanup in preparation to be an accepting vector. Concentration is 4.0 ng/uL.
Trial Two:
Figure Two: Nanodrop of new pNIC-Bsa4 in DH5alpha after PCR cleanup in preparation to be an accepting vector. Concentration is 49.9 ng/uL.
Analysis: In this experimen the solution made from midiprep was used. The pNIC-Bsa4 was cut using NEB Buffer 4, 100x BSA, and BsaI-HF in order to prepare pNIC-Bsa4 as an accepting vector that can accept T. brucei protein phosphatase 2B, putative for cloning. In trial one all of the sample (pNIC-Bsa4) from midiprep trial one was lost during PCR clean up or was cut wrong resulting in such a low concentration. If the concentration of pNIC-Bsa4 goes down a little after cutting and clean up, this is okay because the clean up is removing any contamination, but in trial one too much of the pNIC-Bsa4 was lost so we could not proceed to cloning. In trial two only about 5 ng/uL was lsot after cutting and clean up so this loss was contamination and the trial two of pNIC-Bsa4 (cut) is ready to proceed to cloning. Nothing was changed procedure wise during transformation, midiprep, and cutting for trial two only different plates of pNIC-Bsa4 was used and a new PCR clean up kit was used.
Cohesive End Generation, Transformation and Annealing Trial One:
Objective: The purpose of this lab is to prepare insert and accepting vector for successful cloning.
Figure One: T. brucei protein phosphatase 2B, putative with pNIC-Bsa4 on an LB+KAN (no suc) plate after overnight in the incubator.
Analysis: Although there were colonies on the plates both 1:2 and 1:3 ratios of vector to insert, LB+Kan+Suc plates were not used so pNIC-Bsa4 also grew with our DNA because sucrose kills pNIC. But there were some big colonies so putting a variety of colonies on a master plate with sucrose should show if our DNA grew.
Master Plate:
Objective: The purpose of making a master plate is to join insert and accepting vector together to later get positive matches from DNA sequencing.
Figure One: T. brucei protein phosphatase 2B, putative with accepting vector on a master plate with LB+Kan+Suc, grown overnight in the incubator.
Analysis: On the plate rows 1 and 3 is the 1:2 ratio alternating between big colony and little colony and on rows 2 and 4 the ratio 1:3 plates was used also alternating between big colony and little colony. It is good that the larger colonies grew on the plates because the smaller colonies from the first round of plates were supposed to be pNIC-Bsa4 because sucrose plates weren't used and the larger colonies were supposed to be our DNA. Because colonies grew the next step is to send the transformed tubes to DNA sequencing to check for matches. (When all eight tubes were spun down only the four that grew colonies formed a pellet because the other four didn't have any DNA).
Nanodrop check of Transformed Colonies:
Figure One: Nanodrop of T. brucei protein phosphate 2B, putative from colony #1 on master plate. Concentration of colony #1 is 154.6 ng/uL.
Figure Two: Nanodrop of T. brucei protein phosphate 2B, putative from colony #3 on master plate. Concentration of colony #3 is 157.2 ng/uL.
Figure Three: Nanodrop of T. brucei protein phosphate 2B, putative from colony #5 on master plate. Concentration of colony #5 is 159.3 ng/uL.
Figure Four: Nanodrop of T. brucei protein phosphate 2B, putative from colony #7 on master plate. Concentration of colony #7 is 161.2 ng/uL.
Then prepared these solutions and send to DNA sequencing. Waiting for Results
Gel Extraction:
Objective: The purpose of gel extraction is to reduce contamination of DNA by extracting from gel.
Figure One: Gel Check of T. brucei protein phosphatase 2B, putative of PCR squared (from secondary PCR at 63 degC for annealing temperature) to be used for gel extraction.
After gel extraction:
Figure Two: Nanodrop of half of DNA extracted from agarose gel. Concentration of DNA is 75.6 ng/uL.
Figure Three: Nanodrop of the other half of DNA extracted from agarose gel. Concentration of DNA is 73.3 ng/uL.
Analysis: After gel extraction (process where bands were cut from gel and purified using kit) the concentration of our DNA was determined using nanodrop to be around 74 ng/uL which is good value for the concentration to be proceeding into cloning. Because the graph of the DNA concentration of 73.3 ng/uL looks better this will be used as the insert for cloning trial two.
Cloning Trial Two:
Figure One: T. brucei protein phosphatase 2B, putative with accepting vector (pNIC-Bsa 4) on LB+ Kan+Suc plate after overnight incubation.
Analysis: Cloning round two did not work. The same accepting vector made two days prior was used and the new gel extracted DNA was used to make a new insert. The same steps were followed and this time was grown overnight on the correct plates with sucrose. The plates might not have grown because the plates could have been made wrong, the vector could have gone bad, the insert might not have worked properly or the ratios weren't correct. Now I wait for the results or proceed to next round of cloning with new plates, new insert and new vector with different ratios.
OMG this took forever to read... I'm glad after the 3rd trial it worked though :)! I'm loving the detailed analyses and figure captions. Keep up the groundbreaking research! - Michael T.
Weeks 5 and 6
Secondary PCR trials one through three done during week 5 :(
Objective: The purposed of secondary PCR is to take 1 uL of primary PCR that worked properly and PCR again with tail primers in order to run on a gel and get a thick band (with no contamination) the size of the target protein and later use for PCR squared.
Ladders used during primary and secondary PCR: 100 bp ladder and 1 kb ladder
Trial One:
(not sure why this image got distorted when placing on the wikipage)
Figure One: Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. In lane two the 1 kb ladder was used (we really should have used the 100 bp ladder), in lane three secondary PCR run with the primary PCR that was run for longer in the PCR machine. Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane seven contains 1 kb ladder and lane eight contains the secondary PCR with the primary PCR that was run for the shorter amount of time, also containing the same solutions.
Analysis:
Sadly secondary PCR did not work. A faint smear showed up in the wells kind of looking as if it was primary PCR but instead most likely contained contamination or the primers did not attach fully causing the DNA to not cut in the right spot. By using the 1 kb ladder we could not really tell if the protein was the correct size to begin with so next round we used the correct 100 bp ladder. Also we realized that we did not dilute the dNTP which could have also resulted in the secondary PCR to not work. Overall the time and the temperature needs to be changed that way the primers are able to latch onto DNA like they should.
Trial Two:
Figure Two: Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains a 100 bp ladder and lane three contains secondary PCR with the primary PCR that was run for longer in the PCR machine. Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane seven contains 100 bp ladder and lane eight contains the secondary PCR with the primary PCR that was run for the shorter amount of time, also containing the same solutions.
Analysis:
Secondary PCR once again did not work. The main reason that the secondary PCR did not work was that once the samples were run in the PCR machine and we went to retrieve the samples the machine was not closed all the way during the entire run. This being said the samples were not properly run so the primers could not attach to the DNA and the DNA also did not replicate. The times and temperatures were not changed for the second trial of secondary PCR. Overall the time and the temperature needs to be changed and played with that way the primers are able to latch onto DNA like they should.
Trial Three (getting closer!)
Figure Three: Primary and Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains the 100 bp ladder, lane three contains primary PCR (new - containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart). Lane four contains secondary PCR run at a longer annealing time (30 seconds). Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane six contains 100 bp ladder, lane seven contains primary PCR and lane eight contains secondary PCR.
Analysis:
Secondary PCR did not work again but getting on the right track! Primary PCR work perfectly, there is a bright smear starting where it should be and the primary PCR was run at the longer of the times given by the NEB guidelines. The secondary PCR on the other hand did not work again, but this time I changed the annealing time to 30 seconds that way the primers could attach themselves better the DNA/protein. There is a fant band where the size of the protein is but there is a smear in the lanes containing secondary PCR, this may have been caused by not all of the primers attaching. Next the temperature for annealing will be changed by either increasing or decreasing it a couple of degrees to see if the primers full attach themselves and thus produce a bright thick band the size of the protein.
Secondary PCR week six trials 4 - 9
Figure Four: Primary and Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains the 100 bp ladder, lane three contains primary PCR (new - containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart). Lane four contains secondary PCR run at a longer annealing time (30 seconds). Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane six contains 100 bp ladder, lane seven contains primary PCR and lane eight contains secondary PCR.
Analysis: Secondary PCR once again did not work. This time he annealing time was at 30 seconds and one solution was run at a lower annealing temperature of 55 degC and the other at a higher temperature of 62 degC. Nothing really showed up at the lower temperature but the high temperature shows some promise because there is a faint band. Secondary PCR might have worked if the primary turned out better because it was not even at the correct size because it should have been lower in the well. I was not happy with the way this gel turned out because it was pulled out and stopped several times so I ran another gel with Keely's and mine.
Figure Five: Primary and Secondary PCR of T. brucei protein phosphatase 2B, putative on an agarose gel. Lane two contains the 100 bp ladder, lane three contains a new primary PCR (containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart). Lane four contains an old PCR used in the first primary PCR. Lane five contains secondary PCR run at a longer annealing time (30 seconds) and a lower annealing temperature (55 degC). Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane six contains primary PCR and lane eight contains secondary PCR with the annealing time of 30 sec and annealing temperature of 62 degC. Lane seven contains Keely’s secondary PCR at the lower annealing temperature and lane eight contains Keely’s secondary PCR at the higher annealing temperature.
Analysis: Different gel with the same solutions made for previous gel. Once again the gel showed that both the old an new primary PCR did not come out as they should have causing the secondary to not work as it should have. There was once again a faint band with my secondary at the higher temperature but Keely's secondary's produced primer dimer (meaning primers didn't attach). The next step will be to remake primary again, ensure that it works first on a gel before making secondary then make secondary using a temperature gradient on the annealing temperature that is around 62 degC either higher or lower but mostly higher because the targets melting temperature is a lot higher than 58 degC.
Figure Six: Primary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains the 100 bp ladder. Lane three and four contains my primary PCR (containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart) with nothing changed with the NEB guidelines but all the times are at the highest. Lane five and six contain Keely’s primary PCR the same settings as mine.
Analysis: Primary PCR is no longer working like it should. It is too concentrated in one area of the smear which is causing a band to appear where the size of our DNA is. I did not change anything from the first time I ran primary PCR and it worked so this is slightly weird. The fact that the primary is too concentrated and bright might be caused by the oligo mix going bad. But we decided to run the secondary just incase because the primary is so concentrated it should have caused the secondary to work but it still did not. By the results of this primary an oligo mix needs to be remade that way the mix is fresh and will cause the primary and secondary to work.
Figure Seven: Secondary PCR of T. brucei protein phosphatase 2B, putative on an agarose gel. Lane two contains a 1 kb ladder (because 100 bp ladder was out). Lane three contains secondary PCR at the annealing temperature of 65 degC. Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane four contains secondary PCR at the annealing temperature of 64.5. Lane five contains secondary PCR run at the annealing temperature of 63 degC. Lane six contains secondary PCR run at the annealing temperature of 61.5 degC. All secondary PCR’s other times and temperatures were kept at the NEB guidelines.
Analysis: Yet again secondary PCR did not work. Lane six and lane five show some promise because a small band did appear but it is not as concentrated as it should be this might be caused by the fact that primary is so concentrated causing secondary to not be as concentrated as it needs to be. This secondary was made from the primary PCR shown in the previous gel so I did not expect secondary to work. Now I need to start completely over by making the oligo mix and seeing if this causes the primary to work and if I get a good primary then by moving onto secondary and trying different temperatures I should get a secondary PCR that works (hopefully).
Figure Eight: Primary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains 100 bp ladder. Lane three contains my primary PCR (with new oligo mix) containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart with the same settings as alsways. Lane four contains Keely’s primary PCR same as lane three. Lane four contains my primary PCR with 0.5 uL of oligo mix and 0.25 uL of Q5 hotstart. Lane five contains Keely’s primary PCR with half the oligo mix and half the Q5.
Analysis: Primary PCR was redone with a new oligo mix and still resulted in the primary PCR as too concentrated but not as bad as with the older oligo mix, the high concentration is spread throughout the smear more. But once the amount of oligo added was halved and the Q5 was halved my primary PCR produced better results than it has been producing. Keely’s primary PCRs are off though neither one of hers produced smears like mine did which is weird because we ran them on the same machine so she must have done something different with her actually PCR solution that produced these results. Both my primary PCR’s seem okay so the next step will be to take my primary PCR’s and run secondary using different annealing temperatures to see if secondary finally works.
Figure Nine: Secondary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two contains a 100 bp ladder. Lane three contains secondary PCR with primary PCR halved at 62 degC annealing temperature. Secondary PCR sample contains primary PCR, polymerase, forward and reverse primers, diluted dNTP, dH20 and Q5 hotstart. Lane four contains secondary PCR with normal primary PCR at 62 degC. Lane five contains secondary PCR with halved primary PCR at 63 degC. Lane six contains secondary PCR with normal primary PCR at 63 degC. Lane seven contains secondary PCR with halved primary PCR at 64 degC. Lane eight contains secondary PCR with normal primary PCR at 64 degC. Lane nine contains secondary PCR with halved primary PCR at 61 degC. Lane ten contains secondary PCR with normal primary PCR at 61 degC. All other times and temperatures remained the same.
Analysis: Secondary PCR finally worked (even though there is some contamination). All eight sample proved to be successful because finally a bring band appeared where our DNA size is; this is a new 100 bp ladder so it looks like it is the wrong size but its not because the ladder is lower. This time secondary PCR was made with the new primary with the new oligo mix. Halve of the secondary PCRs where made with the normal primary PCR that contained 1 uL of oligo and 0.5 uL of Q5 and the other half of the secondary PCRs contained the primary that had been halved (0.5 uL of oligo and 0.25 uL of Q5). Four were made from each set (either normal or halved primary) at the annealing temperatures of 61, 62, 63, or 64 degC and run at normal NEB guidelines for everything else. But it seems like the secondary PCR worked regardless of the normal or halved primary used, which means that the oligo mixed made at the beginning was not working properly. Now that we have lots of secondary that worked we can move on to PCR squared then curing African sleeping sickness.
*Also done on 10.4.13 and 10.5.13 we grew the overnight culture of pNIC-bsa4.
Week 3 and 4
Nicole - great work!. Dr. B 092713
ps - HF = high fidelity
PCR Trial Two and RE Digest (completed together)
Objective for PCR: The purpose of this experiment is to make a master mix also known as a coding sequence to make into a gel for analysis.
Objective for RE Digest: The purpose of this experiment was to digest pGBR22 plasmid (180.98 ng/uL) with restrictive enzymes EcoRI-HF and PvuII-HF and the buffer NEBuffer 4 and visualize the fragments on the agarose gel.
Figure One: Both RE Digest and PCR were run on this gel. Lanes 2-6 were RE Digest and Lanes 1, 7-10 were PCR. In this gel the RE Digest did not work as planned Lane 5's solution was not made correctly EcoRI-HF must have been added when PvuII-HF should have been added. Lane 2 contains 5 uL of 1kg DNA ladder, Lane 3 contains 12 uL of uncut plasmid (pGBR22 - 187.9 ng/uL), Lane 4 contains 30 uL of sample one with EcoRI-HF, Lane 5 contains 30 uL of sample two with PvuII-HF, and Lane 6 contains 30 uL of sample three with both EcoRI-HF and PvuII-HF (buffer used was NEBuffer 4). PCR did work as planned even though there was some contamination! Lane 1 contains 100bp DNA Ladder/marker, Lane 7 contains sample A with 0.0613 uL of plasmid (pGBR22 - 48.95 ng/uL), 5 uL of master mix, 1 uL of Taq and rest nanopure; Lane 8 contains sample B with .613 uL of plasmid, 5 uL mix, 1 uL Taq, rest nanopure; Lane 9 contains sample C with 6.13 uL plasmid, 5 uL mix, 1 uL Taq, rest nanopure; and Lane 10 contains no plasmid, 5 uL mix, 1 uL Taq, rest nanopure.
Figure Two: Both RE Digest and PCR were run on this gel. Lanes 1-6 were RE Digest and Lanes 7-10 were PCR. In this gel the RE Digest did work as planned. Lane one and two contain 5 uL of 1kg DNA ladder, Lane 3 contains 12 uL of uncut plasmid, Lane 4 contains 30 uL of sample one with EcoRI-HF, Lane 5 contains 30 uL of sample two with PvuII-HF, and Lane 6 contains 30 uL of sample three with both EcoRI-HF and PvuII-HF (buffer used was NEBuffer 4). PCR did work as planned even though there was some contamination! Lane 7 contains 100bp DNA Ladder/marker, Lane 8 contains sample A with 0.0613 uL of plasmid (pGBR22 - 108.98 ng/uL), 5 uL of master mix, 1 uL of Taq and rest nanopure; Lane 9 contains sample B with .613 uL of plasmid, 5 uL mix, 1 uL Taq, rest nanopure; Lane 10 contains sample C with 6.13 uL plasmid, 5 uL mix, 1 uL Taq, rest nanopure; and if there were a Lane 11 it would have contained no plasmid, 5 uL mix, 1 uL Taq, rest nanopure (but it was not put into the gel because we knew nothing would show up considering there was no plasmid in the sample).
Questions on RE Digest protocol:
The appropriate buffer to use for EcoRI-HF and PvuII-HF was NEBuffer 4. We used high frequency of the the two restrictive enzymes so we had to use that specific buffer, but it would have been a different buffer if did not use HF. The temperature recommend for incubation is between 65 degrees Celsius and 80 degrees Celsius. The conditions to stop EcoRI-HF buffer is 65 degrees Celsius for 20 minutes and the conditions to stop PuvII-HF buffer is 80 degrees Celsius for 20 minutes.
Analysis for PCR:
The experiment was done over again with two different trials. The only difference between trial one and trial two was the anneal temperature. In trial one the anneal temperature was set to 42 degC while trial two stayed at the 45 degC listed on the protocol. We did this to see if it would produce different outcomes, but it did not that we are aware of because both of the trails worked. PCR worked this time because the master mix was made correctly, everything was kept at the correct temperature, Taq was added last and the samples were immediately put into the PCR machine unlike last time where the Taq was left out for a while before being put into the solutions. There was some contamination that showed up in the gel, but the thicker lines are at the correct size according the the 100 kb ladder.
Analysis for RE Digest:
In this experiment six samples were made two with EcoRI-HF, two with PvuII-HF and two with both along with the buffer NEBuffer 4 and nanopure. These samples were incubated at 37 degC for 2 hours, spun down, put on a heat block for 20 minutes between the temperatures of 65 degC and 80 degC and spun down again. After this a gel was made with a 1kb DNA ladder, uncut plasmid (but a different plasmid was used not the same as the plasmid used to make the samples because the plasmid was used up; uncut plasmid used was pGBR22 187.9 ng/ul) and the three samples (all done on two different gels). The trial one gel did not work because one of the samples was made incorrectly, I believe I accidentally put the same restrictive enzyme (EcoRI-HF) in both samples one and two. But the trial two experiment was done perfectly and the sizes are correct for each of the samples.
Future:
Both of the experiment are extremely important in the future when both of the processes will have to repeated again with our target. RE Digest is especially important when we will want to cut the plasmid and find what can bind/fit into those cuts.
Primer Overlap (with Q5 Polymerase)
Objective: Thaw out primer to then mix each cell by pipetting up and down then remove 1 uL of plasmid from each cell to add with autoclaved water, then freeze for further research. .
Analysis:
In this experiment a primer was made using 1 uL of primer from each cell. Since T. brucei protein phosphatase 2B, putative had 36 cells 74 uL of autoclaved water was added to the tube to give the solution a total volume of 100 uL. Once completed the sample was put into the -20 degC freezer. This experiment was not been completed but will be in the future.
PCR Primer Design Tails for pNIC-Bsa4 Cloning
Objective: The purpose of this lab was to design the forward and reverse primer of T. brucei protein phosphatase 2B, putative and insert in pNIC-Bsa4.
Coding Sequence for T. Brucei protein phosphatase 2B, putative
TACTTCCAATCCATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGATGGTCTCAAAGAACGTGAATACGTTTGGAAAAAATCTCACGCGGACGAGTCCCCGG
GTTCTAACCCGCAAATCCTGCCGACCCTGGTTCTGCCGCACCACCTCGTGTTCGACAACGACGGTGCGCCACTGGCGGACAACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCTCCACGTTGAGGATGCGCTGAATATCGTTCACCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCTGCAGTAGTCTGCGGTGATCTCC
ACGGCCAGTTCCACGACCTCCTGACCCTGCTGGAAGTTAATGGTCACCCTAGCGTTCAACAGTACGTTTTCCTGGGTGACTACGTTGACCGTGGTGACTTCTCTGCTGAAA
TCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCCGCGCTCTTTCATCCTGCTGCGTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAGGAAAT
CGAATCTAAATACTCTTCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTGTCTTGCGTTGTTAACGACCGTTTCTTTTGTGTTCATGGTGGTCTCTCTCC
GCTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGTGAAACCCCGTCTACTGGTCCGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACC
GACTGTGCGACGCCGAGCGAAGAACTGTTCGTTTTTAACACTAAACGTGGTTGCTCTTACAACTACAGCTACGAAGCCGTTTGCCGTTTCCTCGAAGCGAACAATCTGTGCA
CGGTTATTCGTGGTCATGAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTGTTCCGGCGGTTGTATGTGTTTTTAGCGCGAGCAATTATTGCGGTAC
CTACGGTAACATGGCCGCAGTTGTAGCGATCGATGGTGACGTTATGAACATCCGTCAGTACATGGCGACCTCTCACGACTCTTGCACCCCTAACCACTTCAATGCGATCTC
TCGTATGCAGCCTCTGGCGATCCATGAGGCGGTAGAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAAAAAGTTGAGGTTGAAAAAAACCT
GTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGCGATATGATCTGCGCAATGCACCACATTGTTACCCAGTAAAGGTGGATA
Forward Primer (33 base pairs)
TACTTCCAATCCATGTCTTCTGGCGCGTCCCAT (order)
Melting Temperature
0 mM Mg 66.8 degC
1.5 mM Mg 73.6 degC
2 mM Mg 74.1 degC
4 mM Mg 75.0 degC
6 mM Mg 75.4 degC
Reverse Primer (38 base pairs)
GCGCAATGCACCACATTGTTACCCAGTAAAGGTGGATA
Reverse complement
TATCCACCTTTACTGGGTAACAATGTGGTGCATTGCGC (order)
Melting Temperature
0 mM Mg 66.4 degC
1.5 mM Mg 73.7 degC
2 mM Mg 74.1 degC
4 mM Mg 74.9 degC
6 mM Mg 75.3 degC
Final Sequence with Coding Sequence in pNIC
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGATGGTCTCAAAGAACGTGAATACGTTTGGA
AAAAATCTCACGCGGACGAGTCCCCGGGTTCTAACCCGCAAATCCTGCCGACCCTGGTTCTGCCGCACCACCTCGTGTTCGACAACGACGGTGCGCCACTGGCGGACAA
CATCAAAGTTCACTTCGGTCGCGGTTGGCGCCTCCACGTTGAGGATGCGCTGAATATCGTTCACCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAA
GGGCTCTGCAGTAGTCTGCGGTGATCTCCACGGCCAGTTCCACGACCTCCTGACCCTGCTGGAAGTTAATGGTCACCCTAGCGTTCAACAGTACGTTTTCCTGGGTGACTA
CGTTGACCGTGGTGACTTCTCTGCTGAAATCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCCGCGCTCTTTCATCCTGCTGCGTGGTAACCACGAGTCTCGCCAGCTG
ACGTCTTGCTTCAACTTCAAACAGGAAATCGAATCTAAATACTCTTCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTGTCTTGCGTTGTTAACGACCG
TTTCTTTTGTGTTCATGGTGGTCTCTCTCCGCTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGTGAAACCCCGTCTACTGGTCCGATGTGTGACCTGCTCT
GGTCTGATCCAATGTTCGGTGATGACACCGACTGTGCGACGCCGAGCGAAGAACTGTTCGTTTTTAACACTAAACGTGGTTGCTCTTACAACTACAGCTACGAAGCCGTTTG
CCGTTTCCTCGAAGCGAACAATCTGTGCACGGTTATTCGTGGTCATGAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTGTTCCGGCGGTTGTATG
TGTTTTTAGCGCGAGCAATTATTGCGGTACCTACGGTAACATGGCCGCAGTTGTAGCGATCGATGGTGACGTTATGAACATCCGTCAGTACATGGCGACCTCTCACGACTCT
TGCACCCCTAACCACTTCAATGCGATCTCTCGTATGCAGCCTCTGGCGATCCATGAGGCGGTAGAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAA
GCGGAAAAAGTTGAGGTTGAAAAAAACCTGTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGCGATATGATCTGCGCAATGCACCACATTGTTACCCAGTAAA
GGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCT
GAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAAT
GGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC
TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAG
GGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA
ACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACG
TTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCA
TCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATG
GCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGA
CTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATT
CGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTT
TCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAA
GAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATA
CAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCC
CGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC
CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC
TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC
TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC
GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGG
AGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCC
ACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG
CTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAG
TGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCA
TAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGC
ATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGT
CGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTT
TTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACAT
GCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGT
GTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCAT
TCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCG
GGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCT
TGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACC
TGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGG
TCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCA
ACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGC
AGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAG
ATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCA
TTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGAC
GCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATA
CTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGC
CCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAG
ATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA
ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATAC
TCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCG
GGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGG
CGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGA
TATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGGATCTCGATCCCGCGAAAT
Figure One: Virtual gel of CDS sequence of T. brucei protein phosphatase 2B, putative with enzyme BsaI.
Figure Two: Virtual gel of pNIC28-Bsa4 sequence with enzyme BsaI.
Figure Three: Virtual gel of CDS of T. brucei protein phosphatase 2B, putative in pNIC28-Bsa4 with enzyme BsaI.
Analysis:
In this experiment the coding sequence for T. brucei protein phosphatase 2B, putative was used along with upstream and downstream primers for LIC cloning to make forward and reverse primers to be ordered for later experimentation. The coding sequence was added to pNIC-Bsa4 to create a final sequence with no SacB gene. In order to get the melting temperatures the same between the forward and reverse primers more amino acids (bases) were added to the reverse that way when they were both at 2mM Mg (and other standards) that the melting temperatures were both 74.1 degC. 33 base pairs for forward primer and 38 base pairs for reverse primer were needed to have the same melting temperatures. The reverse complement will be ordered later because the primer will read oppositely which is why the reverse must be ordered. After ordering the next step will be to see what can fit into the SacB gene of out target's plasmid.
Primary PCR
Objective: The purpose of this lab was to make the primary PCR solution with our target's diluted template/primer to see if it showed up on an agarose gel.
Figure One: Primary PCR of T. brucei protein phosphatase 2B, putative on agarose gel. Lane two has the 1kg DNA ladder and lane three has the primary PCR containing reaction buffer, diluted dNTP, dH2O, template and Q5 hotstart. Lane three is different than lane eight because it was run in the PCR machine for the longer of the NEB recommended guidelines for time while lane eight had the shorter cycle times. Lane seven contains 1kg DNA ladder and lane eight contains the primary PCR solution at the shorter cycle time.
Figure Two: 1kg Ladder used in gel above.
Analysis:
In this experiment a primary PCR solution was made using 5X rxn buffer, diluted dNTP, our oligo mix, dH2O and Q5 hotstart polymerase and run through the PCR machine with the NEB recommend guidelines. One solution was run at the higher ends of cycle time and the other solution was run at the lower end of the cycle times listed. The solutions remained in the PCR machine overnight then were moved to the -20 degC freeze before running the gel. The gel showed that the primary PCR did work because there is a smear in that well where the solution was put in; in lane three the smear was around 1.8 kb which is the size of our target. The primary PCR that was run for the longer time did show up better than the one that ran for a shorter time, but both worked. The next step is the do the secondary PCR with the forward and reverse primers ordered earlier this week, but we are waiting for the primers to come in in order to start the next step.
Week 1 and 2
Nicole, great job. Crop your agarose gel images (can do in the software) to remove alot of the black. Re-do PCR this week.. Dr. B 090913
Nanodrop Spectorphotometer: Determine Concentration of Plasmids, DNA
Objective:
The purpose of this experiment was to relearn how to use the nanodrop spectrophotometer for future experiments and to properly take measurements of pGBR22.
Images:
Figure One: Plot of pGBR22 (205.4 ng/uL) using the nanodrop spectrophotometer that found the concentration to be 202.8 ng/uL.
Figure Two: Plot of pGBR22 (205.4 ng/uL) using the nanodrop spectrophotometer that found the concentration to be 206.8 ng/uL.
Results/Conclusion:
This experiment refreshed and reinforced how to properly take measurements utilizing the nanodrop spectrophotometer. pGBR22's original concentration is 205.4 ng/uL. The first measurement found the concentration to be 202.8 ng/uL with a 260/280 value of 1.98 and a 260/230 value to be 1.83, which is close to the original concentration value. The second measurement found the concentration to be 206.8 ng/uL with a 260/280 value of1.94 and a 260/230 value to be 1.81. The second concentration value was also close to the original concentration value therefore both measurements were valid. The average of the two concentrations measured was found to be 204.8 ng/uL. Errors could have been made during the experiment if the machine was not properly blanked skewing future data or some of the water may not have been wiped off before the first measurement was taken causing the concentration to dilute a small amount possibly accounting for the reason that the first measurements concentration was lower than the second.
Nanodrop Spectrophotometer: Determine Concentration of Plasmid
Objective:
The purpose of this lab was to prepare a plasmid for the core lab for them to add a primer to the solution for later analysis.
In this experiment option A was chosen.
Images:
Figure One: Documented proof that a request was sent using core website requesting for in-house primer M13F to be add and DNA sequencing to be done on the prepared template of DNA (pGBR22).
Figure Two: Results of the DNA sequencing on the DNA template + primer (M13F) done at the core lab.
Figure Three: Nucleotide BLAST of the coding sequence against Human and added all nucleotide collection databases.
Results/Conclusion:
In this experiment a DNA template using pGBR22 was made and taken to a different lab after the proper request form was sent in. The primer chosen was M13F and the template was taken to the lab the next day after making. Results were emailed several hours later, the analysis of these results will take place in the Analyzing DNA Sequence lab. Since option A was chosen for the experiment several errors could have occurred at the other lab that we have no control over such as using the wrong primer, not adding the correct amount, maybe the primer was not kept at the right temperature, this error also could have occurred in our lab as well if the pGBR22 was not kept at the right temperature during preparation.
PCR Primer Design for Primer Overlap Assembly PCR:
Objective:
The purpose of this experiment was to design an oligo set of primers to be order for further experimentation involving cloning.
-------------------------------------------------------------------------------- | Helix Systems -- Center for Information Technology | | http://helix.nih.gov | | National Institutes of Health, Department of Health and Human Services | | | | DNAWorks Web Site: http://helixweb.nih.gov/dnaworks | -------------------------------------------------------------------------------- | | | Send all correspondence to webtools@helix.nih.gov | -------------------------------------------------------------------------------- Job started on 09/03/2013 at 14:40:38 Job name: NW9022013TBP2B Output will be sent to nicole_welch23@yahoo.com SEQUENCE 1: PROTEIN LENGTH = 431 ---------------------------------------------------------------- 1 MSSGASHHELTRGRDGLKEREYVWKKSHADESPGSNPQILPTLVLPHHLVFDNDGAPLAD 61 NIKVHFGRGWRLHVEDALNIVHRCALIMKEEPNVVRLKGSAVVCGDLHGQFHDLLTLLEV 121 NGHPSVQQYVFLGDYVDRGDFSAEIVLLCMSFKLLYPRSFILLRGNHESRQLTSCFNFKQ 181 EIESKYSSMVYEEIMAAFDCFPLSCVVNDRFFCVHGGLSPLLTYLGEIDTVNRFRETPST 241 GPMCDLLWSDPMFGDDTDCATPSEELFVFNTKRGCSYNYSYEAVCRFLEANNLCTVIRGH 301 ETQPGGYKLYRHTPKGVPAVVCVFSASNYCGTYGNMAAVVAIDGDVMNIRQYMATSHDSC 361 TPNHFNAISRMQPLAIHEAVEKWCVASHGGSSGDAKAEKVEVEKNLSEDDSSVVLREKLG 421 DMICAMHHIVT ---------------------------------------------------------------- ---------------------------------------------------------------- None found ---------------------------------------------------------------- 32 oligonucleotides need to be synthesized ---------------------------------------------------------------- 1 ATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGAT 45 2 CCGCGTGAGATTTTTTCCAAACGTATTCACGTTCTTTGAGACCATCACGACCACGGGTCA 60 3 TTTGGAAAAAATCTCACGCGGACGAGTCCCCGGGTTCTAACCCGCAAATCCTGCCGACCC 60 4 GGCGCACCGTCGTTGTCGAACACGAGGTGGTGCGGCAGAACCAGGGTCGGCAGGATTTGC 60 5 CAACGACGGTGCGCCACTGGCGGACAACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCT 60 6 GATCAGCGCGCAACGGTGAACGATATTCAGCGCATCCTCAACGTGGAGGCGCCAACCGCG 60 7 CCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCTGCAGT 60 8 GTCAGGAGGTCGTGGAACTGGCCGTGGAGATCACCGCAGACTACTGCAGAGCCCTTCAGG 60 9 AGTTCCACGACCTCCTGACCCTGCTGGAAGTTAATGGTCACCCTAGCGTTCAACAGTACG 60 10 GCAGAGAAGTCACCACGGTCAACGTAGTCACCCAGGAAAACGTACTGTTGAACGCTAGGG 60 11 CCGTGGTGACTTCTCTGCTGAAATCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCC 60 12 CGAGACTCGTGGTTACCACGCAGCAGGATGAAAGAGCGCGGGTACAGCAGTTTGAAAGAC 60 13 GTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAGGAAATCGAAT 60 14 CCATGATTTCTTCGTAAACCATAGAAGAGTATTTAGATTCGATTTCCTGTTTGAAGTTGA 60 15 TCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTGTCTTGCGTTGTT 60 16 CAGCGGAGAGAGACCACCATGAACACAAAAGAAACGGTCGTTAACAACGCAAGACAGCGG 60 17 GGTGGTCTCTCTCCGCTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGT 60 18 CAGAGCAGGTCACACATCGGACCAGTAGACGGGGTTTCACGAAAACGGTTAACAGTATCG 60 19 CGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACCGACTGTGCGACGC 60 20 AGAGCAACCACGTTTAGTGTTAAAAACGAACAGTTCTTCGCTCGGCGTCGCACAGTCGGT 60 21 AACACTAAACGTGGTTGCTCTTACAACTACAGCTACGAAGCCGTTTGCCGTTTCCTCGAA 60 22 TGGGTCTCATGACCACGAATAACCGTGCACAGATTGTTCGCTTCGAGGAAACGGCAAACG 60 23 TTCGTGGTCATGAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTG 60 24 CGCAATAATTGCTCGCGCTAAAAACACATACAACCGCCGGAACACCCTTTGGGGTATGGC 60 25 GCGCGAGCAATTATTGCGGTACCTACGGTAACATGGCCGCAGTTGTAGCGATCGATGGTG 60 26 TCGTGAGAGGTCGCCATGTACTGACGGATGTTCATAACGTCACCATCGATCGCTACAACT 60 27 CATGGCGACCTCTCACGACTCTTGCACCCCTAACCACTTCAATGCGATCTCTCGTATGCA 60 28 ACACACCACTTTTCTACCGCCTCATGGATCGCCAGAGGCTGCATACGAGAGATCGCATTG 60 29 GCGGTAGAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAA 60 30 CAGAGCTGTCATCTTCAGACAGGTTTTTTTCAACCTCAACTTTTTCCGCTTTCGCGTCAC 60 31 TGTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGCGATATGATCTGCGCAA 60 32 GGTAACAATGTGGTGCATTGCGCAGATCATATCGCC 36 FINAL SUMMARY FOR 1 SOLUTION -------------------------------------------------------------------------------- # Tm Len | Score TmRange Short Long #Olig #Repeat #Misprime 1 62 60 | 0.000 1.9 14 60 32 0 0Figure One: Final results logfile of T. brucei protein phosphatase 2B, putative using the amino acid sequence found on the TDR website.Protein Length for T. brucei protein phosphatase 2B, putative = 431
Results/Conclusion:
In this experiment the amino acid sequence for T. brucei protein phosphatase 2B, putative was used to find oligoneleotides (primers) to be order for further experiments regarding this target. An error occurred during this experiment because a wrong YP number was given on the Wiki for this target so the experiment was redone with the correct amino acid sequence. Once this problem was fixed the correct oligos were found and are ready to be ordered.
PCR
Objective: The purpose of this experiment is to make a master mix also known as a coding sequence to make into a gel for analysis.
Figure One: Agarose gel with 1kb DNA ladder showing in the second lane. In lane three sample A a 1:1000 dilution template, lane four sample B a 1:1000 dilution differing in the amount of DDW, lane give sample C a 1:100 dilution and lane six sample D with no DNA (control sample). Only the ladder is visible. The picture was taken in the UV imaging box.
Analysis/Conclusions:
In this experiment sample A-D were made differing in the amount of DNA template, template dilution and diluted Taq, and were run in the PCR machine. Once finished in the machine the agarose gel was made but proved to be unsuccessful because only the ladder showed up in the image, and at least one of the samples should have shown up. Several errors could have been made such as the right amounts were not added into each sample, the dilutions were made incorrectly, certain solutions and the samples were not kept at the correct temperature or the gel did not work by expressing the DNA like it should have.
Analyzing DNA Sequencing
Objective: The purpose of this lab was to BLAST sequences and compare sequences in order to determine the DNA sequence of a plasmid.
Figure One: Screenshot of Analyzing DNA Sequencing that is one the Google Drive.
Results/Conclusions:
In this experiment a primer sequence was used and altered in order to make a final reverse complement sequence with part of the original sequence in it. The final sequence was put into NEB where the primer was placed in the primer ring where a gel could be found.